CN108456703B - Method for heterogeneously expressing epothilone - Google Patents

Abstract

The invention discloses a method for heterologously expressing epothilone, which is characterized in that an epothilone gene cluster is introduced into host bacteria, an epothilone precursor synthesis way is supplemented, the introduction of related tRNA genes and the insertion of promoters are combined, the expression quantity of the epothilone can be greatly improved, the yield is improved by about 4 orders of magnitude and can reach 8.5 mg/L. In addition, the invention also provides a genetic engineering strain for heterologously expressing the epothilone and a method for producing the epothilone.

Description

Method for heterogeneously expressing epothilone

Technical Field

The invention relates to the field of biological expression, in particular to a method for heterologously expressing epothilone and a related genetic engineering strain.

Background

Epothilone (epothilones) is produced by fermentation of Sorangium cellulosum, and is a novel antitumor drug similar to taxol (paclitaxel) and having microtubule stabilizing effect. There are 5 epothilones and derivatives currently in clinical use, of which Patupilone (epothilone B) and KOS-862 (epothilone D) are fermentation products, Ixabepilone and BMS-310705 are chemical modifiers of epothilone B, and ZK-EPO is a chemically fully synthetic drug. Ixabepilone was approved by the FDA in the united states for advanced breast cancer treatment in 10 months of 2007. At present, the main fermentation strain of epothilone is cellulose sorangium which is the primary strain, and natural strains mainly produce epothilone A and B.

Polysaccharum brevicornum [ Polyangium ] brachysporum DSM7029 (═ K481-B101 ═ ATCC 53080) was isolated from soil in 1988, and DSM7029 strain produced taxin, an antifungal and antitumor drug, as a proteasome inhibitor, a natural product of the hybrid NRPS/PKS (non-ribosomal polypeptide synthase/polyketide synthase) type. The strain DSM7029 has not yet been assigned a classification status and belongs to the order Burkholderia (Burkholderia) by 16S rDNA analysis. The growth speed of the strain is faster than that of the original bacterium cellulose sorangium cellulosum and the model Myxococcus xanthus which are produced by the epothilone, and a single colony can be seen on two days. The strain is found to heterologously express the epothilone.

Through the prior art, the originally produced bacteria cellulose sorangium cellulosum can reach about 100mg/L after being mutagenized and optimized, but the fermentation period exceeds 20 days, so the method is easy to pollute and hardly has space for improving. And other model bacteria such as escherichia coli or non-modified DSM7029 can not express the epothilone efficiently, and the yield can only reach about 1 ug/L. Therefore, there is a need to develop a method for heterologous expression of epothilones with high efficiency and genetically engineered strains for production.

Disclosure of Invention

In order to overcome the problem of low expression efficiency of epothilone in the prior art, one aspect of the invention provides a method for heterologously expressing epothilone. In one embodiment, the method introduces an epothilone gene cluster in the host bacterium, while complementing the epothilone precursor synthesis pathway. Wherein the epothilone gene cluster is derived from sorangium cellulosum.

Further, the host bacteria belong to the order Burkholderia (Burkholderia).

Further, the host bacterium is a strain of Burkholderia DSM 7029.

Further, the precursor synthesis pathway is that of S-methylmalonyl-CoA.

Further, the synthetic pathway that supplements the S-methylmalonyl-coa is one or more of a PCC pathway, a MatB pathway, and a mutase-isomerase pathway.

Preferably, the synthesis pathway that complements said S-methylmalonyl-coa is the complement of the PCC pathway, the MatB pathway and the mutase-isomerase pathway.

Further, the PCC pathway is complemented by the addition of propionyl-CoA carboxylase (propionyl-CoA carboxylase); the MatB pathway is supplemented by the addition of malonyl-CoA/methylmalonyl-CoA synthetase (malonyl-CoA/methylmalonyl-CoA synthase); the mutase-isomerase pathway is complemented by the addition of methylmalonyl-CoA isomerase (methylmalonyl-CoA isomerase).

Further, propionyl-CoA carboxylase is the accA1/pccB or pccA/pccB gene of Streptomyces coelicolor A3 (2).

Further, methylmalonyl-coa isomerase is the epi gene of streptomyces coelicolor a3 (2).

Further, malonyl-coa/methylmalonyl-coa synthetase is the matB gene of streptomyces coelicolor a3 (2).

Further, tRNA genes are introduced into the host bacterium.

Further, the tRNA gene is one or more of Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes.

Further, Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes were derived from Myxococcus xanthus DK 1622.

Further, a promoter sequence is added in front of one or more genes in the epothilone gene cluster.

Preferably, promoter sequences are added pre-to one or more of the epoA, epoB, epoC, epoD, epoE and epoF 6 genes in the epothilone gene cluster.

Further preferably, a promoter sequence is added before each of the epoA, epoB, epoC, epoD, epoE and epoF 6 genes in the epothilone gene cluster.

Further, the promoter is PKan.

Further, promoter sequences were added by splicing genes anew.

Further, splicing was performed using the Bxb1 integrase splicing technique.

In another aspect of the invention, a genetically engineered strain for heterologous expression of epothilones is provided. In one embodiment, the genetically engineered strain incorporates an epothilone gene cluster supplemented with an epothilone precursor synthesis pathway. Wherein the epothilone gene cluster is derived from sorangium cellulosum.

Further, the basic strain of the genetically engineered strain was the Burkholderia (Burkholderia) DSM7029 strain.

Further, the basic strain is supplemented with the synthetic pathway of epothilone precursor S-methylmalonyl-coenzyme A.

Further, the synthetic pathway of S-methylmalonyl-coa includes the PCC pathway, MatB pathway, and mutase-isomerase pathway.

Further, in the base strain, the PCC pathway was supplemented by the addition of accA1/pccB from streptomyces coelicolor (s. coelicolor) a3 (2); supplementation of the mutase-isomerase pathway by addition of the epi gene of streptomyces coelicolor a3 (2); the matB pathway was complemented by the addition of the matB gene of streptomyces coelicolor a3 (2).

Further, tRNA genes were added to the base strain.

Further, the tRNA gene is Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC gene.

Furthermore, a promoter sequence is added in front of one or more genes of the epothilone gene cluster.

Preferably, a promoter sequence is added to each of the epoA, epoB, epoC, epoD, epoE and epoF 6 genes in the epothilone gene cluster.

Further, the promoter sequence is added by splicing in order of epoA, epoB, epoC, epoD, epoE to epoF by means of re-splicing.

Further, the promoter is PKan.

Further, in a preferred embodiment of the present invention, the genetically engineered strain for heterologous expression of epothilone is Polysaccharum brevicompactum (Polyangium brachysporum) MMR11 with a collection number of CCTCC NO: M2017037, which is collected in the China center for type culture Collection on 19.1.2017, address: wuhan university in Wuchang Lojia mountain in Wuhan city, Hubei province.

In yet another aspect, the invention provides a method for producing an epothilone. In a specific embodiment, it provides a strain as described above, fermented in a fermentation medium at 30 ± 2 ℃.

Further, the fermentation medium is CYMG fermentation medium, the formula of each liter is 8g of casein peptone, 4g of yeast extract, 4.06g of magnesium chloride hexahydrate, 10ml of 50% glycerol, 1ml of trace elements, 50mg of sodium acetate, 100mg of sodium propionate, 100mg of methylmalonic acid, 2.5mg of cysteine, 5mg of serine and 1% of wet weight of XAD-16 macroporous adsorption resin, and the pH is adjusted to be 7.0-7.5.

Further, the fermentation time was 3 days.

The method for heterologous expression of the epothilone improves the yield of the epothilone by analyzing the host strain genome and the epothilone gene cluster, modifying the synthesis route of an epothilone precursor S-methylmalonyl coenzyme A lacking in the host strain and expression elements such as tRNA and promoter suitable for the epothilone gene cluster. In a preferred embodiment, the epothilone is efficiently expressed in the modified DSM7029 strain, the yield is improved by about 4 orders of magnitude, reaches 8.5mg/L, is 105 times of that of the epothilone produced by the strain only containing the unmodified gene cluster, and meets the requirement of industrial production of the epothilone.

Drawings

FIG. 1 is a complementary schematic representation of the synthesis pathway of the precursor S-methylmalonyl-CoA in one embodiment of the present invention.

FIG. 2 is a graph of the precursor S-methylmalonyl-CoA synthesis pathway and epothilone yield following tRNA addition in an embodiment of the invention.

FIG. 3 shows a peak transcriptome profile of the epothilone gene cluster of strain MMR1 containing the epothilone gene cluster (T is the sense strand and F is the antisense strand) in an embodiment of the present invention.

FIG. 4 is a schematic representation of the reassembling and promoter addition of the epothilone synthesis gene cluster in an embodiment of the present invention.

FIG. 5 is a map of the plasmid pST-BSD-epo in one embodiment of the present invention.

FIG. 6 is a graph showing the epothilone yield following the rejoining of the epothilone biosynthetic gene cluster in an embodiment of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the present invention.

The invention conjectures the reason of low yield of the heterologous expression epothilone in the conventional host strain by genome analysis of the host strain and analysis of the epothilone gene cluster, and modifies the host strain aiming at the factors, thereby achieving the method for improving the heterologous expression of the epothilone and providing related modified strains.

DSM7029 strain characterization and epothilone gene cluster characterization

The genome of DSM7029 was not previously sequenced and sequencing and analysis of the genome of DSM7029 revealed a GC content of 67.51% in the genome of DSM7029, which is close to 69.5% in the cluster of 56kb epothilone.

The DSM7029 genome comprises multiple NRPSs and PKSs, with the largest CDS encoding non-ribosomal peptide synthase (AAW51 — 3371) of 32,469bp in size. Whereas the epothilone gene cluster derived from Sorangium cellulosum encodes a hybrid NRPS/PKS of 56kb in size, comprising 9 PKS modules, 1 NRPS module and 1P 450 oxidase. The complete epothilone gene cluster comprises 7 genes of epoA (shown in SEQ ID No. 1), epoB (shown in SEQ ID No. 2), epoC (shown in SEQ ID No. 3), epoD (shown in SEQ ID No. 4), epoE (shown in SEQ ID No. 5), epoF (shown in SEQ ID No. 6) and epoK. Wherein, P450 oxidase (EpoK) can catalyze epothilone C \ D to form an epoxy structure and convert to epothilone A \ B. Epothilones C and D were produced with only 6 epoA, epoB, epoC, epoD, epoE and epoF genes. The epothilone gene cluster derived from Sorangium cellulosum lacks the necessary promoter sequence for expression in DSM 7029.

When the epothilone is produced, the epothilone gene cluster derived from Sorangium cellulosum can be selected, and the epothilone gene cluster derived from other strains can also be selected.

Analysis of tRNA:

from the tRNA statistics and codon preference analysis of epothilones for DSM7029 strain (using tRNAscan-SE v1.23 analysis), DSM7029 strain lacks 4 trnas that mainly express epothilones: arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC. Comparison of the tRNA from DSM7029 strain with Myxococcus xanthus, Sorangium cellulosum (see Table 1) revealed that three of the above tRNAs were similarly deficient. Supplementation with tRNAs was significant for epothilone production.

Table 1 comparison of tRNA species and amounts in M.xanthus DK1622, S.cellulosum So0157-2, and DSM7029 strains

-: absence of

Analysis of precursor synthesis pathway:

the epothilone gene cluster is a PKS/NRPS type gene cluster, and the synthesis precursor of epothilone A comprises 1 molecule of acetate, 4 molecules of S-methylmalonyl coenzyme A, 4 molecules of malonyl coenzyme A, one molecule of SAM-derived methyl carbon and one molecule of cysteine. When epothilone B is synthesized, one molecule of malonyl-CoA is replaced by methylmalonyl-CoA. Among them, S-methylmalonyl-coenzyme A is an important synthetic precursor of epothilone.

S-methylmalonyl-coenzyme A has the following synthetic pathway:

1) PCC route: propionyl-CoA synthetase (propionyl-CoA, prpE, EC:6.2.1.17) catalyzes the synthesis of propionyl-CoA, followed by the synthesis of S-methylmalonyl-CoA ((2S) -methylmalonyl-CoA) by propionyl-CoA carboxylase (propionyl-CoA carboxylase, pccA/pccB, EC: 6.4.1.3);

2) MatB pathway: the substrate methylmalonate methyl-CoA (methylmalonyl-CoA) is synthesized by malonyl-CoA/methylmalonyl-CoA synthetase (malonyl-CoA/methylmalonyl-CoA synthase, matB, EC:6.2.1. -);

3) mutase-isomerase (mutase-epimerase) pathway: succinyl-CoA is converted to R-methylmalonyl-CoA ((2R) -methylmalonyl-CoA) by methylmalonyl-CoA mutase (methylmalonyl-CoA mutase, mcMA, EC:5.4.99.2), and S-methylmalonyl-CoA ((2S) -methylmalonyl-CoA) is produced by methylmalonyl-CoA isomerase (methylmalonyl-CoA epimerase, epi, 5.1.99.1).

Annotated alignments of the genome of strain DSM7029 found that strain DSM7029 lacks the complete S-methylmalonyl-coa synthesis pathway, which lacks the PCC pathway and the mutase-isomerase pathway.

Based on the above analysis, modifications from the point of view of supplementing the epothilone precursor synthesis pathway, addition of appropriate tRNA and addition of promoter are contemplated to increase epothilone yield.

The present invention will be further explained with reference to specific examples.

The operations in the examples may be carried out by conventional techniques in the art unless otherwise specified.

Restriction enzymes were purchased from Thermo Fisher Scientific; ex Taq, GC buffer I/II, Primerstar^TMHS DNA polymerase, lambda-EcoT 14Marker was purchased from Takara-Bio engineering (Dalian) Ltd; the DNA agarose gel recovery kit and the bacterial genome extraction kit are purchased from Shanghai Czeri organisms; the PCR product recovery kit is purchased from biological engineering; cloneexpress MultiS, Cloneexpress II, Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novozam; XAD-16 (macroporous adsorbent resin) was purchased from Shanghai Momo scientific apparatus; epothilone B was purchased from Toronto Research Chemicals; epothilone a, epothilone C, epothilone D were purchased from a reconnect melphalan organism; chromatographic grade methanol and chromatographic grade acetonitrile were purchased from MERCK, germany; ultimate XB-C18,5 μm, 4.6X 250mm, available from Yuekang science and technology (Shanghai); casein peptone was purchased from BD corporation, usa; yeast extract was purchased from OXIOD; magnesium chloride and glycerol are purchased from biological engineering.

Antibiotics:

example 1 addition of precursor metabolic pathway genes and tRNA's to increase epothilone production

As analyzed previously, the DSM7029 strain lacks the complete S-methylmalonyl-coa synthesis pathway, which lacks the PCC pathway and the mutase-isomerase pathway. Thus, the pathway of S-methylmalonyl-CoA synthesis was considered to be supplemented to increase epothilone production, and the supplemented pathway is shown in FIG. 1. The operation mode is that firstly, a plasmid containing the epo gene cluster is constructed and transferred into the DSM7029 strain, then plasmids containing different complementary pathway genes and/tRNA genes are constructed and transferred into the DSM7029 strain containing the epo gene cluster, and a modified strain is obtained.

Plasmid 1 (containing the epo gene cluster) was constructed as follows: plasmids Cosmid 10 and Fosmid3B11 (see Allopatric integrations selected change host transactions, leading to varied expression of exogenous genes in Myxococcus xanthus, Microb Cell Fact [ J ]., 2015; 14:105.) obtained by screening the Sorangium cellulosum genome So0157-2 library, respectively, contained a 38.5kb fragment from epoA to the front of the epoD and a 34.4kb fragment from the back of the epoC to the downstream of the epoF fragment of the epothilone biosynthetic gene cluster, both fragments having a 6.5kb region coverage. PCR targeting fragment 1(SEQ ID No.7) and fragment 2(SEQ ID No.8) were synthesized in a whole gene (wherein the p15A replicon, the resistance gene and att site were partially located on the completely synthesized fragment 1 and fragment 2), and reacted with plasmids Cosmid 10 and Fosmid3B11, respectively, using PCR targeting technology, to obtain plasmids pZLE21 (containing attB0 and attP6 sites) and pZLE22 (containing attB6 and attP15 sites). Finally, the three pZLE21, pZLE22 and pZLE19 (containing attB15 and attP0 sites) were reacted using phi BT1 integrase (plasmid pZLE19 and integrase reactions referenced to the Tandem assay of the epothilone biochemical gene cluster by in vitro site-specific recombination, Sci Rep.2011; 1:141.doi:10.1038/srep00141) to give plasmid pZL-epo containing the entire epothilone biosynthesis gene cluster. Wherein, the attB0 site is shown as SEQ ID No.9, the attP6 site is shown as SEQ ID No.12, the attB6 site is shown as SEQ ID No.11, and the attP15 site is shown as SEQ ID No. 18.

Plasmid pZL-epo containing the epothilone Gene cluster is transformed into GBred strain (purchased from Gene Bridges), electroporation competence is prepared, primers BSD-epo-F/R (shown as SEQ ID Nos. 71 and 72) are utilized, plasmid pBSD (refer to Nucleic Acids Res.2008Oct; 36(17): e113.doi: 10.1093/nar/35499, which can be inserted into DSM7029 genome through transposition) containing transposase is used as a template, an amplification product is electroporated into GBred/pZL-epo, and plasmid pBSD-epo containing transposase and the complete epothilone Gene cluster can be obtained through homologous recombination and is named as plasmid 1.

The PCC pathway was supplemented by the addition of the accA1/pccB or pccA/pccB gene from Streptomyces coelicolor A3(2) (purchased from ATCC, accession number ATCC BAA-471) according to the supply pathway of S-methylmalonyl-CoA; an epi gene derived from Streptomyces coelicolor A3(2) is added to complement a mutase-isomerase pathway; the addition of the matB gene from Streptomyces coelicolor A3(2) increased the number of copies that expressed another malonyl-CoA/methylmalonyl-CoA synthase.

Specifically, a streptomyces coelicolor genome is taken as a template, an accA1-F/R (shown as SEQ ID Nos. 21 and 22) is taken as a primer to amplify an accA1 gene (shown as SEQ ID No. 31), a pccA-F/R (shown as SEQ ID Nos. 23 and 24) is taken as a primer to amplify a pccA gene (shown as SEQ ID No. 32), and a pccB-F/R (shown as SEQ ID Nos. 25 and 26) is taken as a primer to amplify a pccB gene (shown as SEQ ID No. 33); epi-F/R (shown as SEQ ID Nos. 27 and 28) is used as a primer to amplify epi gene (shown as SEQ ID No. 34); MatB-F/R (shown as SEQ ID No.29 and 30) is used as a primer to amplify the matB gene (shown as SEQ ID No. 35).

Plasmid pBSD is used as a template, BSD-F/R (shown as SEQ ID No.19 and 20) is used as a primer to amplify a vector fragment, and the vector fragment is connected with the genes by using a seamless connection technology.

As analyzed previously, the DSM7029 strain lacks 4 trnas that predominantly express epothilones: arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC. Therefore, it is considered to increase these 4 tRNAs to improve the yield of epothilone.

Four tRNA derived from myxococcus aurantiacus are synthesized in a whole gene, the Arg anti-GCG sequence is shown as SEQ ID No.36, the Arg anti-TCG sequence is shown as SEQ ID No.37, the Gln anti-CTG sequence is shown as SEQ ID No.38, the Glu anti-CTC sequence is shown as SEQ ID No.39, the whole gene synthesis tRNA sequence of the 4 genes is shown as SEQ ID No.70, and tRNA-F/R (shown as SEQ ID Nos. 40 and 41) is used as a primer for cloning.

After a promoter PKan (SEQ ID No. 42) was added to each of the 5 amplified genes in the precursor metabolic pathway and to the entire tRNA gene (in the entire gene synthesis, an entire fragment was synthesized in the order of Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC), the fragment was constructed into a transposase-containing vector pBSD by the seamless ligation technique. Wherein, 7 plasmids are constructed, which are respectively: plasmid 2(pccA + pccB), plasmid 3(pccA + pccB + tRNA), plasmid 4(accA1+ pccB), plasmid 5(accA1+ pccB + tRNA), plasmid 6(accA1+ pccB + epi + tRNA), plasmid 7(accA1+ pccB + matB + tRNA), plasmid 8(accA1+ pccB + epi + matB + tRNA) to see the effect of different additive combinations on epothilone production. The constructed plasmid 1 is converted into DSM7029 to obtain recombinant bacteria MMR1 containing epothilone gene clusters, and the plasmids 2-8 are taken to be respectively converted into MMR1 to obtain various epothilone expression recombinant bacteria MMR 2-8. The specific transformation process is as follows: adding 3 microliters of plasmid to be transformed into DSM7029 or MMR1 competent cells transferred into an epothilone gene cluster, uniformly mixing, adding a 2mm electric shock cup, performing electric transformation at 2500V, and coating a plate containing kalamycin (Kanamycin) or Apramycin (Apramycin) after activating for 3 hours. Positive clones were picked on plates after 2 days.

The fermentation of epothilone uses 500ml CYMG fermentation medium, the formula is 8g of casein peptone (Casitone) (BD company), 4g of yeast extract (OXOID company), 4.06g of magnesium chloride hexahydrate, 10ml of 50% glycerol, 1ml of trace elements, 50mg/L of sodium acetate, 100mg/L of sodium propionate, 100mg/L of methylmalonic acid, 2.5mg/L of cysteine, 5mg/L of serine, 1% of XAD-16 macroporous adsorption resin wet weight, water is added to 1L, the pH is adjusted to 7.0-7.5, and moist heat sterilization is carried out for 20min at 121 ℃. The fermentation temperature is 30 ℃, the rotating speed of a shaking table is 200rpm, and the fermentation lasts for 3 days.

The trace elements are: 0.79g of manganese chloride tetrahydrate, 0.15g of zinc sulfate heptahydrate, 0.64g of copper sulfate pentahydrate and 0.11g of ferrous sulfate heptahydrate are dissolved in 100mL of water and used as mother liquor for later use.

After fermentation, the resin was poured into a 100 mesh standard sieve and washed several times, dried and then added with 25ml of methanol, and the solution was resolved twice at 30 ℃ for 12 hours each time. And concentrating the methanol analysis solutions of the two times, uniformly mixing, filtering, and quantifying the yield of the epothilone A, B, C and D by UHPLC-MS/MS.

As shown in FIG. 2, the original strain DSM7029 had no epothilone production. The recombinant strain MMR1 is obtained by adding an epothilone gene cluster (epo gene cluster) into DSM7029, and the yield of epothilone C and D reaches 61.27 mu g/L and 18.76 mu g/L. On the basis, a pccA/pccB/tRNAs path is supplemented to obtain a strain MMR3, and the total yield is improved by 10%; on the basis of the supplement of the accA1/pccB and accA1/pccB/tRNAs pathways, the strains MMR4 and MMR5 are obtained, the total yield is doubled and is 100 percent and 130 percent respectively, and the yield of epothilone C and D reaches 129.54 mu g/L and 59.35 mu g/L. The strains MMR6 and MMR7 obtained by respectively supplementing the epi gene and the MatB gene on the basis of supplementing the accA1/pccB/tRNAs pathway have continuously and greatly increased epothilone yield, and the epothilone yield A, B, C and D reaches 65.57 mu g/L, 58.35 mu g/L, 508.30 mu g/L, 466.40 mu g/L, 19.35 mu g/L, 3.39 mu g/L, 225.20 mu g/L and 47.33 mu g/L. When all the approaches of accA1-pccB-tRNAs-epi-matB are supplemented, the strain MMR8 is obtained, the yield reaches the highest, the yields of epothilone A, B, C and D reach 45.85 mug/L, 62.54 mug/L, 399.12 mug/L and 1101.03 mug/L, the total yield reaches 1.6mg/L, and is 20 times of the yield of the strain only added with the epo gene cluster.

Example 2 Re-splicing of the epo Gene Cluster to add promoters to increase epothilone production

After transcriptome sequencing is carried out on the strain MMR1 containing the epothilone gene cluster, the expression level of each gene in the epothilone gene cluster is very low (figure 3), and the low-level gene expression can be another key factor for limiting the yield of the epothilone.

In view of the above, to improve the expression efficiency of the epothilone synthetic gene cluster in DSM7029, the 6 genes of the epothilone gene cluster epoA, epoB, epoC, epoD, epoE and epoF were re-spliced as shown in FIG. 4 using the Bxb1 integrase splicing technique. Promoters are added in front of each gene in the epothilone gene cluster to increase the expression quantity of each gene in the gene cluster. Attempts were made to increase epothilone production by adding promoters.

Specifically, plasmid pSB1A3 (from iGEM, website http:// parts.org/Part: pSB1A3) is used as a template, epo1A3-F/R (shown in SEQ ID Nos. 43 and 44) is used as a primer for cloning Amp sequence, plasmid pSB3K5 (from iGEM, website http:// parts.org/Part: pSB3K5) is used as a template, epo3K5-F/R (shown in SEQ ID Nos. 45 and 46) is used as a primer for cloning P15A sequence, and the two fragments are connected in a seamless manner to obtain plasmid P-vector (containing four restriction enzyme cutting sites of EcoRI, XbaI, SpeI and PciI). Cloning epoA, pEpoB, pEpoC and pEpoF by taking a plasmid pZL-epo containing an epothilone gene cluster as a template, cloning the epoA gene by taking the epoA-F/R (shown as SEQ ID Nos. 47 and 48) as a primer, cloning the epoB gene by taking the epoB-F/R (shown as SEQ ID Nos. 49 and 50) as a primer, cloning the epoC gene by taking the epoC-F/R (shown as SEQ ID Nos. 51 and 52) as a primer, cloning the epoF gene by taking the epoF-F/R (shown as SEQ ID Nos. 57 and 58) as a primer, amplifying a vector fragment by taking the epoF-F/R (shown as SEQ ID Nos. 73 and 74) as a primer and taking the p-vector as a template, and cloning the genes in a seamless connection mode to obtain the pEpoA, pEpoB, pEpoC and pEpoF.

Transferring the plasmid pZL-epo containing the epothilone gene cluster into a GBred strain to prepare electrotransformation competence, amplifying intermediate vector linearization fragments containing homologous arms by using an intermediate vector p-vector as a template and epoD-F/R (shown as SEQ ID Nos. 53 and 54) and epoE-F/R (shown as SEQ ID Nos. 55 and 56) as primers, then electrotransfering into the GBred/pZL-epo strain, and obtaining plasmids pEpoD and pEpoE containing epoD and epoE through homologous recombination.

The plasmids pEpoA, pEpoB, pEpoC, pEpoD, pEpoE and pEpoF containing the epothilone gene were digested with restriction enzymes EcoRI and XbaI. pBSD is used as a template, a primer pKan-F/R (shown as SEQ ID No.59 and 60) is used as a primer amplification promoter sequence, the PKan promoter is subjected to double enzyme digestion by using restriction enzymes EcoRI and SpeI, and an enzyme digestion fragment is recovered. And (3) connecting the double-enzyme-digested epothilone gene and the PKan promoter fragment to obtain epothilone gene plasmids pPKan-EpoA, pPKan-EpoB, pPKan-EpoC, pPKan-EpoD, pPKan-EpoE and pPKan-EpoF added with the promoter.

The plasmid pPKan-EpoB is cut by restriction enzymes EcoRI and XbaI, the plasmid pPKan-EpoA is cut by restriction enzymes EcoRI and SpeI, and the pPKan-EpoA-PKan-EpoB is obtained after ligation. The plasmid pPKan-EpoF is cut by restriction enzymes EcoRI and XbaI, the plasmid pPKan-EpoE is cut by restriction enzymes EcoRI and SpeI, and the pPKan-EpoF is obtained after ligation. The plasmid pPKan-EpoC is cut by restriction enzymes EcoRI and XbaI, the plasmid pPKan-EpoA-PKan-EpoB is cut by restriction enzymes EcoRI and SpeI, and the pPKan-EpoA-PKan-EpoB-pPKan-EpoC is obtained after ligation. The plasmids pPKan-EpoA-PKan-EpoB-pPKan-EpoC, pPKan-EpoD and pPKan-EpoE-PKan-EpoF were digested with restriction enzymes EcoRI and XbaI, respectively, to be ligated to attB0 (shown in SEQ ID No. 9), attB13 (shown in SEQ ID No. 15) and attB7 (shown in SEQ ID No. 13) sites, respectively, to give plasmids pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC, pattB13-PKan-EpoD and pattB 7-PKan-EpoE-PKan-EpoF. The plasmids, pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC, pattB13-PKan-EpoD and pattB7-PKan-EpoE-PKan-EpoF, were digested with the restriction enzymes SpeI and PciI to join, respectively, the attP13 (shown in SEQ ID No. 16), attP7 (shown in SEQ ID No. 14) and attP15 (shown in SEQ ID No. 18) sites to give pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC-attP13, pattB13-PKan-EpoD-attP7 and pattB7-PKan-EpoE-PKan-EpoF-attP 15.

Synthesizing an attP0-ccdB-attB15 fragment (shown as SEQ ID No. 61) in a whole gene mode, taking a synthetic sequence as a template, and taking ccdB-F/R (shown as SEQ ID Nos. 62 and 63) as a primer amplification fragment; the pBSD is used as a template, a primer ccdB-vector-F/R (shown as SEQ ID No.64 and 65) is used for amplifying a fragment, and the two are connected by seamless cloning to obtain a plasmid pBSD-ccdB. Taking pBSD-ccdB as a template and taking a primer BSD-ccdB-F/R (shown as SEQ ID Nos. 66 and 67) as a primer to amplify a fragment; the plasmid pST-ccdB is used as a template, a primer ST-F/R (shown as SEQ ID No.68 and 69) is used for amplifying a fragment, and the two are connected in a seamless mode to obtain the plasmid pST-BSD.

Taking 0.5 microliter of vector plasmid pST-BSD, 2 microliter of pattern B0-PKan-EpoA-PKan-EpoB-pPKan-EpoC-attP13 plasmid, 2 microliter of pattern B13-PKan-EpoD-attP7 plasmid, 1.5 microliter of pattern B7-PKan-EpoE-PKan-EpoF-attP15 plasmid, 1 microliter of Bxb1 integrase, reacting for 20 hours at 30 ℃, transforming the reaction system after treating with high temperature and protease K, and screening out correct clones on an antibiotic kanamycin plate. The map of the correctly sequenced plasmid pST-BSD-epo is shown in FIG. 5.

And (3) taking 5 microliters of pST-BSD-epo plasmid to transfer to a DSM7029 strain to obtain a recombinant strain MMR10 containing the modified epothilone gene cluster added with the promoter. And continuously electrotransfering a plasmid containing accA1-pccB-tRNAs-epi-matB on the basis of the strain of the modified gene cluster to obtain the strain MMR11 with high yield of the epothilone.

The strains MMR1, MMR10, MMR8 and MMR11 are fermented for 3 days by using 500mL CYMG fermentation medium at the fermentation temperature of 30 ℃ and the rotation speed of a shaker of 200 rpm.

After fermentation, the resin was collected in a 100 mesh standard sieve and washed several times, dried and then added with 25ml of methanol, and analyzed twice at 30 ℃ for 12 hours each time. And concentrating the methanol analysis solutions of the two times, uniformly mixing, filtering, and quantifying the yield of the epothilones C and D by UHPLC-MS/MS.

As shown in fig. 6, DSM7029 broth was free of epothilone production. After adding the unspliced epothilone gene cluster in DSM7029, the yield of epothilone C and D of strain MMR1 reached 61.27. mu.g/L and 18.76. mu.g/L. When the spliced epothilone gene cluster added with the promoter is transferred into DSM7029, the generation of epothilone can be detected, the yield of epothilone C and D is 83.63 mu g/L and 25.38 mu g/L, and the total amount is improved by 36 percent. When tRNAs and accA1-pccB-epi-matB genes were added to strains containing unmodified epothilone gene clusters, epothilone C and D yields reached 399.12. mu.g/L and 1101.03. mu.g/L. When the tRNAs and the precursor related genes are added into the strain containing the rejoined promoter, the yield is greatly improved, the yields of the epothilones C and D are 4721.47 mu g/L and 3812.25 mu g/L, and the total yield is improved by 105 times compared with the strain only containing the unmodified gene cluster.

In conclusion, the pathways such as PCC pathway, MatB pathway, mutase-isomerase pathway and the like for synthesizing S-methylmalonyl coenzyme A are added into Burkholderia DSM7029, and the pathways are combined with tRNAs and promoter modification which are necessary for expressing an epothilone gene cluster, so that the epothilone is efficiently expressed in DSM7029, the yield of the epothilone in 3 days in a shake flask can reach 8.5mg/L, and the requirement of industrial production of the epothilone is basically met.

Sequence listing

<110> university of Compound Dan

<120> method for heterologously expressing epothilone

<160> 74

<170> PatentIn version 3.5

<210> 1

<211> 4266

<212> DNA

<213> Sorangium cellulosum

<400> 1

atggcggatc gtcccatcga gcgcgcagcc gaagatccga ttgcgatcgt cggagcgggt 60

tgccgtctgc ccggcggcgt gatcgatctg agcgggttct ggacgctcct cgagggctcg 120

cgcgacaccg tcgggcaagt ccccgcagaa cgctgggatg cagcagcgtg gtttgatccc 180

gacctcgatg ccccggggaa gacgcccgtt acgcgcgcat ctttcctgag cgacgtagcc 240

tgcttcgacg cccccttctt cggcatctcg cctcgcgaag cgctgcggat ggaccctgca 300

catcgactct tgctggaggt gtgctgggag gcgctggaga acgccgcgat cgctccatcg 360

gcgctcgtcg gtacggaaac gggagtgttc atcgggatcg gcccgtccga gtatgaggcc 420

gcgctgccgc gagcgacggc gtccgcagag atcgacgctc atggcgggct ggggacgatg 480

cccagcgtcg gagcgggccg aatctcgtat gtcctcgggc tgcgagggcc gtgtgtcgcg 540

gtggatacgg cctattcgtc ctcgctcgtg gccgttcatc tggcctgtca gagcttgcgc 600

tccggggaat gctccacggc cctggctggt ggggtatcgc tgatgttgtc gccgagcacc 660

ctcgtgtggc tctcgaagac ccgcgcgctg gccacggacg gtcgctgcaa ggcgttttcg 720

gcggaggccg atgggttcgg acgaggcgaa gggtgcgccg tcgtggtcct caagcggctc 780

agtggagccc gcgcggacgg cgatcggata ttggcggtga ttcgaggatc cgcgatcaat 840

cacgacggag cgagcagcgg tctgaccgtg ccgaacggga gctcccaaga aatcgtgctg 900

aaacgggccc tggcggacgc aggctgcgcc gcgtcttcgg tgggttatgt cgaggcacac 960

ggcacgggca cgacgcttgg tgaccccatc gaaatccaag cgctgaatgc ggtatacggc 1020

ctcgggcgag acgtcgccac gccgctgctg atcgggtcgg tgaagaccaa ccttggccat 1080

cctgagtatg cgtcggggat cactgggctg ctgaaggtcg tcttggccct tcagcacggg 1140

cagattcctg cgcacctcca cgcgcaggcg ctgaaccccc ggatctcatg gggtgatctt 1200

cggctgaccg tcacgcgcgc ccggacaccg tggccggact ggaatacgcc gcgacgggcg 1260

ggggtgagct cgttcggcat gagcgggacc aacgcgcatg tggtgctgga agaggcgccg 1320

gcggcgacgt gctcaccgcc ggcgccggag cggccggcag agctgctggt gctgtcggca 1380

aggaccgcgg cagccctgga tgcacacgcg gcgcggctgc gcgaccatct ggagacctac 1440

ccttcgcagt gtctgggcga tgtggcgttc agtctggcga cgacgcgcag cgcgatggag 1500

caccggctcg cggtggcggc gacgtcgagc gaggggctgc gggcagccct ggacgctgcg 1560

gcgcagggac agacgccgcc cggtgtggtg cgcggtatcg ccgattcctc acgcggcaag 1620

ctcgcctttc tcttcaccgg acagggggcg cagacgctgg gcatgggccg tgggctgtac 1680

gatgtatggc ccgcgttccg cgaggcgttc gacctgtgcg tgaggctgtt caaccaggag 1740

ctcgatcggc cgctccgcga ggtgatgtgg gccgaaccgg ccagcgtcga cgccgcgctg 1800

ctcgaccaga cagccttcac ccagccggcg ctgttcacct tcgagtatgc gctcgccgcg 1860

ctgtggcggt cgtggggcat agagccggag ttggtcgctg gccatagcat cggtgagctg 1920

gtggctgcct gcgtggcggg cgtgttctcg cttgaggacg cggtgttcct ggtggctgcg 1980

cgcgggcgcc tgatgcaggc gctgccggcc ggcggggcga tggtgtcgat cgcggcgccg 2040

gaggccgatg tggctgctgc ggtggcgccg cacgcagcgt cggtgtcgat cgccgcggtc 2100

aacggtccgg accaggtggt catcgcgggc gccgggcaac ccgtgcatgc gatcgcggcg 2160

gcgatggccg cgcgcggggc gcgaaccaag gcgctccacg tctcgcatgc gttccactca 2220

ccgctcatgg ccccgatgct ggaggcgttc gggcgtgtgg ccgagtcggt gagctaccgg 2280

cggccgtcga tcgtcctggt cagcaatctg agcgggaagg ctggcacaga cgaggtgagc 2340

tcgccgggct attgggtgcg ccacgcgcga gaggtggtgc gcttcgcgga tggagtgaag 2400

gcgctgcacg cggccggtgc gggcaccttc gtcgaggtcg gtccgaaatc gacgctgctc 2460

ggcctggtgc ctgcctgcct gccggacgcc cggccggcgc tgctcgcatc gtcgcgcgct 2520

gggcgtgacg agccagcgac cgtgctcgag gcgctcggcg ggctctgggc cgtcggtggc 2580

ctgttctcct gggccggcct cttcccctca ggggggcggc gggtgccgct gcccacgtac 2640

ccttggcagc gcgagcgcta ctggatcgac acgaaagccg acgacgcggc gcgtggcgac 2700

cgccgtgctc cgggagcggg tcacgacgag gtcgaggagg ggggcgcggt gcgcggcggc 2760

gaccggcgca gcgctcggct cgaccatcca ccgcccgaga gcggacgccg ggagaaggtc 2820

gaggccgccg gcgaccgtcc gttccggctc gagatcgatg agccaggcgt gcttgatcac 2880

ctggtgcttc gggtcacgga gcggcgcgcc cctggtctgg gcgaggtcga gatcgccgtc 2940

gacgcggcgg ggctcagctt caatgatgtc cagctcgcgc tgggcatggt gcccgacgac 3000

ctgccgggaa agcccaaccc tccgctgctg ctcggaggcg agtgcgccgg gcgcatcgtc 3060

gccgtgggcg agggcgtgaa cggccttgtg gtgggccaac cggtcatcgc cctttcggcg 3120

ggagcgtttg ctacccacgt caccacgtcg gctgcgctgg tgctgcctcg gcctcaggcg 3180

ctctcggcga ccgaggcggc cgccatgccc gtcgcgtacc tgacggcatg gtacgcgctc 3240

gacagaatag cccgccttca gccgggggag cgggtgctga tccacgcggc gaccggcggg 3300

gtcggtctcg ccgcggtgca gtgggcgcag cacgtcggag ccgaggtcca tgcgacggcc 3360

ggcacgcccg agaagcgcgc ccacctggag tcgctgggcg tgcggtatgt gagcgattcc 3420

cgctcggacc ggttcgtcgc cgacgtgcgc gcgtggacgg gcggcgaggg agtagacgtc 3480

gtgctcaact cgctttcggg cgagctggtc gacaagagtt tgaatctcct gcgatcgcac 3540

ggccggtttg tggagctcgg caagcgcgac tgttacgcgg ataaccagct cgggctgcgg 3600

ccgttcctgc gcaatctctc cttctcgctg gtggatctcc gggggatgat gctcgagcgg 3660

ccggcgcggg tccgtgcgct cttcgaggag ctcctcggcc tgatcgcggc aggcgtgttc 3720

acccctcccc ccatcgcgac gctcccgatc gctcgtgtcg ccgatgcgtt ccggagcatg 3780

gcgcaggcgc agcatcttgg gaagctcgta ctcacgctgg gtgacccgga cgtccagatc 3840

cgtattccga cccacgcagg cgccggcccg tccaccgggg atcgggacct gctcgacagg 3900

ctcgcgtcag ctgcgccggc cgcgcgcgcg gcggcgctgg aggcgttcct ccgtacgcag 3960

gtctcgcagg tgctgcgcac gcccgaaatc aaggtcggcg cggaggcgct gttcacccgc 4020

ctcggcatgg actcgctcat ggccgtggag ctgcgcaatc gtatcgaggc gagcctcaag 4080

ctgaagctgt cgacgacgtt cctgtccacg tcccccaata tcgccttgtt gacccaaaac 4140

ctgctggatg ctctcgccac agctctctcc ttggagcggg tggcggcgga gaacctacgg 4200

gcaggcgtgc aaagcgactt cgtctcatcg ggcgcagatc aagactggga aatcattgcc 4260

ctatga 4266

<210> 2

<211> 4233

<212> DNA

<213> Sorangium cellulosum

<400> 2

atgacgatca atcagcttct gaacgagctc gagcaccagg gtatcaagct ggcggccgat 60

ggggagcgcc tccagataca ggcccccaag aacgccctga acccgagcct gctcgctcga 120

atctccgagc acaaaagcac gatcctgacg atgctccgtc agagactccc cgcagagtcc 180

atcgtgcccg ccccagccga gcggcacgtt ccgtttcctc tcacagacat ccaaggatcc 240

tactggctgg gtcggacagg agcgtttacg gtccccagcg ggatccacgc ctatcgcgaa 300

tacgactgtg cggatctcga cgtggcgagg ctgagccgcg cctttcggaa agtcgtcgcg 360

cggcacgaca tgcttcgggc ccacacgctg cccgacatga tgcaggtgat cgagcctaaa 420

gtcgacgccg acatcgagat catcgatctg cgcgggctcg accggagcac gcgggaagcg 480

aggctcgtat cgttgcgaga tgcgatgtcg caccgcatct atgacaccga gcgccctccg 540

ctctatcacg tcgtcgccgt tcggctggac gagcggcaaa cccgtctcgt gctcagcatc 600

gatctcatta acgttgacct aggcagcctg tccatcatct tcaaggactg gctcagcttc 660

tacgaagatc ccgagacctc tctccctgtc ctggagctct cgtaccgcga ctatgtactc 720

gcgctggagt ctcgcaagaa gtctgaggcg catcaacgat cgatggatta ctggaagcgg 780

cgcatcgccg agctcccacc tccgccgatg ctcccgatga aggccgatcc atctaccctg 840

aaggagatcc gcttccggca cacggagcaa tggctgccgt cggactcctg gagtcgattg 900

aagcggcgtg tcggggagcg cgggctgacc ccgacgggcg tcatcctggc tgcattttcc 960

gaggtgatcg ggcgctggag cgcgagcccc cggtttacgc tcaacataac gctcttcaac 1020

cggctacccg tccatccgtg cgtgaacgat atcaccgggg acttcacgtc gatggttctc 1080

ctggacatcg acaccactcg cgacaagagc ttcgaacaac gcgctaagtg tattcaaaag 1140

cagctatggg aggcgatgga tcactgcgac gtgagcggta tcgaggtcca gcgagaggcc 1200

gcccgggtcc tggggatcca acgaggcgca ttgttccccg tagtgctcac gagcgcgctc 1260

aaccagcaag tcgtcggtgt cacctcgctg cagaggctcg gcactccggt gtacaccagc 1320

acgcagactc ctcagctgct gctggatcat cagctctacg agcacgatgg ggacctcgtc 1380

ctcgcgtggg acatcgtcga cggagtgttc ccgcccgacc ttctggacga catgctcgaa 1440

gcgtacgtcg ctcttctccg gcggctcact gaggaaccat ggggtgaaca gatgcgctgt 1500

tcgcttccgc ctgcccagct agaagcgcgg gcgagcgcaa acgagaccaa cgcgctgctg 1560

agcgagcata cgctgcacgg cctgttcgcg gcgcgggtcg agcagctgcc tatgcagctc 1620

gccgtggtgt cggcgcgcaa gacgctcacg tacgaagagc tttcgcgccg ttcgcggcga 1680

tttggcgcgc ggctgcgcga gcagggggca cgcccgaaca cattggtcgc ggtggtgatg 1740

gagaaaggct gggagcaggt tgtcgcggtt ctcgcggtgc tcgagtcagg cgcggcctac 1800

gtgccgatcg atgccgacct accggcggag cgtatccact acctcctcga tcatggtgag 1860

gtaaagctcg tgctgacgca gccatggctg gatggtaaac tgtcatggcc gccggggatc 1920

cagcggctgc tcgtgagcga ggccggcgtc gaaggcgacg gcgaccagct tccgatgatg 1980

cccattcaga caccttcgga tctcgcgtat gtcatctaca cctcgggatc cacagggttg 2040

cccaaggggg tgatgatcga tcatcggggt gccgtcaaca ccatcctgga catcaacgag 2100

cgcttcgaaa tagggcccgg agacagagtg ctggcgctct cctcgctgag cttcgatctc 2160

tcggtctatg atgtgttcgg gatcctggcg gcgggcggta cgatcgtggt gccggacgcg 2220

tccaagctgc gcgatccggc gcattgggca gagttgatcg aacgagagaa ggtgacggtg 2280

tggaactcgg tgccggcgct gatgcggatg ctcgtcgagc attccgaggg tcgccccgat 2340

tcgctcgcta ggtctctgcg gctttcgctg ctgagcggcg actggatccc ggtgggcctg 2400

cctggcgagc tccagaccat caggcccggc gtgtcggtga tcagcctggg cggggccacc 2460

gaagcgtcga tctggtccat cgggtaccca gtgatgaacg tcgatccatc gtgggcgagc 2520

atcccctacg gccgtccgct gcgcaaccag acgttccacg tgctcgatga ggcgctcgaa 2580

ccgcgcccgg tctgggttcc ggggcaactc tacattggcg gggtcggact ggcactgggc 2640

tactggcgcg atgaagagaa gacgcgcaag agcttcctcg tacaccccga gaccggggag 2700

cgcctttaca agaccggcga tctgggccgc tacctgcccg atggaaacat cgagttcatg 2760

gggcgggagg acaaccaaat caagcttcgc ggataccgcg ttgagctcgg ggaaatcgag 2820

gaaacgctca agtcgcatcc gaacgtacgc gacgcggtga ttgtgcccgt cgggaacgac 2880

gcggcgaaca agctccttct agcctatgtg gtcccggaag gcacacggag acgcgctgcc 2940

gagcaggacg cgagcctcaa gaccgagcgg atcgacgcga gagcacacgc cgccgaagcg 3000

gacggcttga gcgacggcga gagggtgcag ttcaagctcg ctcgacacgg actccggagg 3060

gatctggacg gaaagcccgt cgtcgatctg accgggctgg ttccgcggga ggcggggctg 3120

gacgtctacg cgcgtcgccg tagcgtccga acgttcctcg aggccccgat tccatttgtt 3180

gagttcggcc gattcctgag ctgcctgagc agcgtggagc ccgacggcgc ggcccttccc 3240

aaattccgtt atccatcggc tggcagcacg tacccggtgc agacctacgc gtacgccaaa 3300

tccggccgca tcgagggcgt ggacgagggc ttctattatt accacccgtt cgagcaccgt 3360

ttgctgaagg tctccgaaca cgggatcgag cgcggagcgc acgttccgca aaacttcgac 3420

gtgttcgatg aagcggcgtt cggtctcctg ttcgtgggca ggatcgatgc catcgagtcg 3480

ctgtatggat cgttgtcacg agagttctgc ctgctggagg ccggatatat ggcgcagctc 3540

ctgatggagc aggcgccttc ctgcaatatc ggcgtctgcc cggtgggtca attcaatttt 3600

gaacaggttc ggccggttct cgacctgcgg cgttcggacg tttacgtgca cggcatgctg 3660

ggtgggcggg tagacccgcg gcagttccag gtctgtacgc tcggtcagga ttcctcaccg 3720

aggcgcgtca cgacgcgcgg cgcccctcct ggccgcgatc agcacttcgc cgatatcctt 3780

cgcgacttct tgaggaccaa actacccgag tatatggtgc ctacagtctt cgtggagctc 3840

gatgcgttgc cgctgacgtc caacggcaag gtcgatcgta aggccctgcg cgagcggaag 3900

gatacctcat cgccgcggca ttcggagcac acggcgccac gggacgcctt ggaggagatc 3960

ctcgtcgcgg tcgtacggga ggtgctcggg ctggaggtgg tcgggctcca gcagagcttc 4020

gtcgatcttg gtgcgacatc gattcacatc gttcgcatga ggagcctgtt gcagaagagg 4080

ctggataggg agatcgccat caccgagttg ttccagtacc cgaacctcgg ctcgctggca 4140

tccggtttgc gccgagactc gaaagatcta gatcagcgga cgaacatgca ggaccgagtg 4200

gaggcccggc gcaagggcag gagacgtagc taa 4233

<210> 3

<211> 5499

<212> DNA

<213> Sorangium cellulosum

<400> 3

atggaagaac aagattcctc cgctatcgca gtcatcggca tgtcgggccg atttccgggg 60

gcgcgtaatc tggacgagtt ctggaggaac cttcgagacg gcacggaggc cgtgcagcgc 120

ttctccgagc aggagctcgc ggcgtccgga gtcgaccccg cgctggtgct ggacccgagc 180

tacgtccggg cgggcagcgt gctggaagat gtcgaccggt tcgacgctgc tttcttcggc 240

atcagcccgc gcgaggcaga gctcatggat ccgcagcacc gcatcttcat ggaatgcgcc 300

tgggaggcgc tggagaacgc cggatacgac ccgacggctt acgagggctc tatcggcgtg 360

tacgccggcg ccaacatgag ctcgtacttg acgtcgaacc tccacgagca cccagcgatg 420

atgcggtggc ccggctggtt tcagacgttg atcggcaacg acaaggatta cctcgcgacc 480

cacgtctcct acaggctgaa tctgagaggg ccgagcatct ccgttcaaac tgcctgctcc 540

acctcgctcg tggcggttca cttggcgtgc atgagcctcc tggaccgcga gtgcgacatg 600

gcgctggccg gcgggattac cgtccggatc ccccatcgag ccggctatgt atatgccgag 660

gggggcatct tctctcccga cggccattgc cgggccttcg acgccaaggc gaacggcacg 720

atcatgggca acggctgcgg ggttgtcctc ctgaagccgc tggaccgggc gctctccgat 780

ggtgatcccg tccgcgcggt catccttggg tctgccacaa acaacgacgg agcgaggaag 840

atcgggttca ctgcgcccag tgaggtgggc caggcgcaag cgatcatgga ggcgctggcg 900

ctggcagggg tcgaggctaa cccatcgatc gatttcgcga cccacgggac cggcacgctg 960

ctcggagacg ccatcgagac ggcggcgttg cggcgggtgt tcgatcgcga cgcttcggcc 1020

cggaggtctt gcgcgatcgg ctccgtgaag accggcatcg gacacctcga atcggcggct 1080

ggcatcgccg gtttcatcaa gacggtcttg gcgctggagc accggcagct gccgcccagc 1140

ctgaacttcg agtctcctaa cccatcgatc gatttcgcga gcagcccgtt ctacgtcaat 1200

acctctctta aggattggaa taccggctcg actccgcggc gggccggcgt cagctcgttc 1260

gggatcggcg gcaccaacgc ccatgtcgtg ctggaggaag cacccgcggc gaagcttcct 1320

gccgcggcgc cggcgcgctc tgccgagctc ttcgtcgtct cggccaagag cgcagcggcg 1380

ctggatgccg cggcggaacg gctacgagat catctgcagg cgcaccaggg gctctcgttg 1440

ggcgacgtcg ccttcagcct ggcgacgacg cgcagcccca tggagcaccg gctcgcgatg 1500

gcggcgccgt cgcgcgaggc gttgcgagag gggctcgacg cagcggcgcg aggccagacc 1560

ccgccgggcg ccgtgcgtgg ccgctgctcc ccaggcaacg tgccgaaggt ggtcttcgtc 1620

tttcccggcc agggctctca gtgggtcggt atgggccgtc agctcctggc tgaggaaccc 1680

gtcttccacg cggcgctctc ggcgtgcgac cgggccatcc aggccgaagc cggttggtcg 1740

ctgctcgccg agctcgccgc cgacgaaggg tcctcccagc tcgagcgcat cgacgtggtg 1800

cagccggtgc tgttcgcgct tgcggtggca cttgcggcgc tgtggcggtc gtggggtgtc 1860

gcgcccgacg tcgtgatcgg ccacagcatg ggcgaggtag ccgccgcgca tgtggccggg 1920

gcgctgtcgc tcgaggatgc ggtggcgatc atctgccggc gcagccggct gctccggcgc 1980

atcagcggtc agggcgagat ggcggtgacc gagctgtcgc tggccgaggc cgagacagcg 2040

ctccgaggct acgaggatcg ggtgagcgtg gccgtgagca acagcccgcg ctcgacggtg 2100

ctctcgggcg agccggcagc gatcggcgag gtgctgtcgt ccctgaacgc gaagggggtg 2160

ttctgccgtc gggtgaaggt ggatgtcgcc agccacagcc cgcaggtcga cccgctgcgc 2220

gaggacctct tggcagccct gggcgggctc cggccgcgtg cggctgcggt gccgatgcgc 2280

tcgacggtga cgggcgccat ggtagcgggc ccggagctcg gagcgaatta ctggatgaac 2340

aacctcaggc agcctgtgcg cttcgccgag gtagtccagg cgcagctcca aggcggccac 2400

ggtctgttcg tggagatgag cccgcatccg atcctaacga cttcggtcga ggagatgcgg 2460

cgcgcggtcc agcgggcggg cgcagcggtg ggctcgctgc ggcgggggca ggacgagcgc 2520

ccggcgatgc tggaggcgct gggcgcgctg tgggcgcagg gctaccctgt accctggggg 2580

cggctgtttc ccgcgggggg gcggcgggta ccgctgccga actatccctg gcagcgcgag 2640

cggtactgga tcgaagcgcc ggccaagagc gccgcgggcg atcgccgcgg cgtgcgtgcg 2700

ggcggtcacc cgctcctcgg tgaaatgcag accctgtcaa cccagacgag cacgcggctg 2760

tgggagacga cgctggatct caagcggctg ccgtggctcg gcgaccaccg ggtgcaggga 2820

gcggtcgtgt ttccgggcgc ggcgtacctg gagatggcga tttcgtcggg ggccgaggct 2880

ttgggcgatg gcccattgca gataactgac gtggtgctcg ccgaggcgct ggccttcgcg 2940

ggcgacgcgg cggtgttggt ccaggtggtg acgacggagc agccgtcggg acggctgcag 3000

ttccagatcg cgagccgggc gccgggcgct ggccacgcgt ccttccgggt ccacgctcgc 3060

ggcgcgttgc tccgagtgga gcgcaccgag gtcccggctg ggcttacgct ttccgctgtg 3120

cgcgcacggc tccaggccag catacccgcc gcggccacct acgcggagct gaccgagatg 3180

gggctgcagt acggccctgc ctttcagggg attgctgagc tatggcgggg tgaaggcgag 3240

gcgctgggac gggtacgcct gcccgacgcg gccggctcgg cagcggagta tcggttgcat 3300

cctgcgctgc tggacgcgtg cttccagatc gtcggcagcc tcttcgccgg cggtggcgag 3360

gcgacgccgt gggtgcccgt ggagttgggc tcgctgcggc tcttgcagcg gccttcgggg 3420

gagctgtggt gccatgcgcg cgtcgtgaac catgggcacc aaacccccga tcggcagggc 3480

gccgactttt gggtggtcga cagctcgggt gcagtggtcg ccgaagtctg cgggctcgtg 3540

gcgcagcggc ttccgggagc ggtgcgccgg cgcgaagaag acgattggtt cctggagctc 3600

gagtgggaac ccgcagcggt cggcacagcc aaggtcaacg cgggccggtg gctgctcctc 3660

ggcggcggcg gtgggctcgg cgccgcgttg cgctcgatgc tggaggccgg cggccatgcc 3720

gtcgtccatg cggcagagaa caacacgagc gctgccggcg tgcgcgcgct cctggcaaag 3780

gcctttggcg gccaggctcc gacggcggtg gtgcacctcg gcagcctcga tgggggtggc 3840

gagctcgacc cagggctcgg ggcgcaaggc gcattggacg cgccccggag cgccgacgtc 3900

agtcccgatg ccctcgatcc ggcgctggta cgtggctgcg acagcgtgct ctggaccgtg 3960

caggccctgg ccggcatggg ctttcgagac gccccgcgat tgtggcttct gacccgcggc 4020

gcacaggccg tcggcgccgg cgacgtctcc gtgacacagg caccgctgct ggggctgggc 4080

cgcgtcatcg ccatggagca cgcggatctg cgctgcgctc gggtcgacct cgatccggcc 4140

cggcccgatg gggagctcgg tgccctgctg gccgagctgc tggccgacga cgccgaagcg 4200

gaagtcgcgt tgcgcggtgg cgagcgatgc gtcgctcgga tcgtccgccg gcagcccgag 4260

acccggcccc gggggaggat cgagagctgc gttccgaccg acgtcaccat ccgcgcggac 4320

agcacctacc tcgtgaccgg cggtctgggt gggctcggtc tgagcgtggc cggatggctg 4380

gccgagcgcg gcgctggtca cctggtgctg gtgggccgct ccggcgcggc gagcgtggag 4440

caacgggcag ccgtcgcggc gctcgaggcc cgcggcgcgc gcgtcaccgt ggcgaaggca 4500

gatgtcgccg atcgggcgca gctcgagcgg atcctccgcg aggttaccac gtcggggatg 4560

ccgctgcggg gcgtcgtcca tgcggccggc atcttggacg acgggctgct gatgcagcag 4620

actcccgcgc ggtttcgtaa ggtgatggcg cccaaggtcc agggggcctt gcacctgcac 4680

gcgttgacgc gcgaagcgcc gctttccttc ttcgtgctgt acgcttcggg agtagggctc 4740

ttgggctcgc cgggccaggg caactacgcc gcggccaaca cgttcctcga cgctctggcg 4800

caccaccgga gggcgcaggg gctgccagcg ttgagcgtcg actggggcct gttcgcggag 4860

gtgggcatgg cggccgcgca ggaagatcgc ggcgcgcggc tggtctcccg cggaatgcgg 4920

agcctcaccc ccgacgaggg gctgtccgct ctggcacggc tgctcgaaag cggccgcgtg 4980

caggtggggg tgatgccggt gaacccgcgg ctgtgggtgg agctgtaccc cgcggcggcg 5040

tcttcgcgaa tgttgtcgcg cctggtgacg gcgcatcgcg cgagcgccgg cgggccagcc 5100

ggggacgggg acctgctccg ccgcctcgct gctgccgagc cgagcgcgcg gagcgcgctc 5160

ctggagccgc tcctccgtgc gcagatctcg caggtgctgc gcctccccga gggcaagatc 5220

gaggtggacg ccccgctcac gagcctgggc atgaactcgc tgatggggct cgagctgcgc 5280

aaccgcatcg aggccatgct gggcatcacc gtaccggcaa cgctgttgtg gacctatccc 5340

acggtggcgg cgctgagcgg gcatctggcg cgggaggcat gcgaagccgc tcctgtggag 5400

tcaccgcaca ccaccgccga ttctgctgtc gagatcgagg agatgtcgca ggacgatctg 5460

acgcagttga tcgcagctaa attcaaggcg cttacatga 5499

<210> 4

<211> 21774

<212> DNA

<213> Sorangium cellulosum

<400> 4

atgactactc gcggtcctac ggcacagcag aatccgctga aacaagcggc catcatcatt 60

cagcggctgg aggagcggct cgctgggctc gcacaggcgg agctggaacg gaccgagccg 120

atcgccatcg tcggtatcgg ctgccgcttc cctggcggcg cggacgctcc ggaagcgttt 180

tgggagctgc tcgacgcgga gcgcgacgcg gtccagccgc tcgacaggcg ctgggcgctg 240

gtaggtgtcg ctcccgtcga ggccgtgccg cactgggcgg ggctgctcac cgagccgata 300

gattgcttcg atgctgcgtt cttcggcatc tcgcctcggg aggcgcgatc gctcgacccg 360

cagcatcgtc tgttgctgga ggtcgcttgg gaggggctcg aggacgccgg tatctcgccc 420

cggtccatcg acgggagccg caccggtgtg ttcgtcggcg ctttcacggc ggactacgcg 480

cgcacggtcg ctcggctgcc gcgcgaggag cgagacgcgt acagcgccac cggcaacatg 540

ctcagcatcg ccgccggacg gctgtcgtac acgctggggc tgcagggacc ttgcctgacc 600

gtcgacacgg cgtgctcgtc atcgctggtg gcgattcacc tcgcctgccg cagcctgcgc 660

gcaggagaga gcgatctcgc gttggcggga ggggtcagcg cgctcctctc ccccgacatg 720

atggaagccg cggcgcgcac gcaagcgctg tcgcccgatg gtcgttgccg gaccttcgat 780

gcttcggcca acgggttcgt ccgtggcgag ggctgtggcc tggtcgtcct caaacggctc 840

tccgacgcgc aacgggatgg cgaccgcatc tgggcgctga tccggggctc ggccatcaac 900

catgatggcc ggtcgaccgg gttgaccgcg cccaacgtgc tggctcagga gacggtcttg 960

cgcgaggcgc tgcggagtgc ccacgtcgaa gctggggccg tcgattacgt cgagacccac 1020

ggcacaggga cctcgctggg cgatcccatc gaggtcgagg cgctgcgggc gacggtgggg 1080

ccggcgcgct ccgacggcac acgctgcgtg ctgggcgcgg tgaagaccaa catcggccat 1140

ctcgaggccg ccgcaggcgt agcgggtctg atcaaggcag cgctttcgct gacgcacgag 1200

cgcatcccgc gaaacctcaa cttccgcacg ctcaatccgc ggatccggct cgagggcagc 1260

gcgctcgcgt tggcgaccga gccggtgccg tggccgcgca cggaccggcc gcgcttcgcg 1320

ggggtgagct cgttcgggat gagcggaacg aacgcgcacg tggtgctgga agaggcgccg 1380

gcggtggagc tggggcctgc cgcgccggag cgctcggcgg agcttctggt gctgtcgggc 1440

aagagcgagg gggcgctcga cgcgcaggcg gcgcggctgc gcgagcacct ggacatgcac 1500

ccggagctcg ggctcgggga cgtggcgttc agcctggcga cgacgcgcag cgcgatgaac 1560

caccggctcg cggtggcggt gacgtcgcgc gaggggctgc tggcggcgct ctcggccgtg 1620

gcgcaggggc agacgccgcc gggggcggcg cgctgcatcg cgagctcgtc gcgcggcaag 1680

ctggcgttgc tgttcaccgg acagggcgcg cagacgccgg gcatgggccg ggggctttgc 1740

gcggcgtggc cagcgttccg ggaggcgttc gaccggtgcg tggcgctgtt cgaccgggag 1800

ctggaccgcc cgctgcgcga ggtgatgtgg gcggaggcgg ggagcgccga gtcgttgttg 1860

ctcgacgaga cggcgttcac ccagcccgcg ctcttcgcgg tggagtacgc gctgacggcg 1920

ctgtggcggt cgtggggcgt agagccggag ctcctggttg ggcatagcat cggggagctg 1980

gtggcggcgt gcgtggcggg ggtgttctcg ctggaagatg gggtgaggct cgtggcggcg 2040

cgcgggcggc tgatgcaggg gctctcggcg ggcggcgcga tggtgtcgct cggcgcgccg 2100

gaggcggagg tggcggcggc ggtggcgccg cacgcggcgc cggtgtcgat cgcggcggtc 2160

aatgggccgg agcaggtggt gatcgcgggc gtggagcgag cggtgcaggc gatcgcggcg 2220

gggttcgcgg cgcgcggcgt gcgcaccaag cggctgcatg tctcgcacgc gttccactcg 2280

ccgctgatgg aaccgatgct ggaggagttc gggcgggtgg cggcgtcggt gacgtaccgg 2340

cggccaagcg tttcgctggt gagcaacctg agcgggaagg tggtcacgga cgagctgagc 2400

gcgccgggct actgggtgcg gcacgtgcgg gaggcggtgc gcttcgcgga cggggtgaag 2460

gcgctgcacg aagccggcgc ggggacgttc gtcgaagtgg gcccgaagcc gacgctgctc 2520

gggctgttgc cagcctgcct gccggagacg gagccgacgc tgctggcgtc gttgcgcgcc 2580

gggcgcgagg aggctgcggg ggtgctcgag gcgctgggcg ggctgtgggc cggcggcggc 2640

tcggtcagct ggccgggcgt cttccccacg gctgggcggc gggtgccgct gccgacctat 2700

ccgtggcagc ggcagcggta ctggatcgag gcgccggccg aagggctcgg agccacggcc 2760

gccgatgcgc tggcgcagtg gttttaccgg gtggactggc ccgagatgcc tcgctcatcc 2820

gtggattcgc ggcgagcccg gtccggcggg tggctggtgc tggccgaccg gggtggagtc 2880

ggggaggcgg ccgcggcggc gctttcgtcg cagggatgtt cgtgcgccgt gctccatgcg 2940

ccggccgagg cctccgcggt cgccgagcag gtgacccagg ccctcggtgg ccgcaacgac 3000

tggcaggggg tgctgtacct ctggggtctg gacgccgtcg tggaggcggg ggcatcggcc 3060

gaagaggtcg gcaaagtcac ccatcttgcc acggcgccgg tgctcgcgct gattcaggcg 3120

ctgggcacgg ggccgcgctc accccggctc tggatcgtga cccgaggggc ctgcacggtg 3180

ggcggcgagc ctgacgctgc cccctgtcag gcggcgctgt ggggtatggg ccgggtcgcg 3240

gcgctggagc atcccggctc ctggggcggg ctcgtggacc tggatccgga ggagagcccg 3300

acggaggtcg aggccctggt ggccgagctg ctttcgccgg acgccgagga tcagctggca 3360

ttccgccagg ggcgccggcg cgcagcgcgg ctcgtggccg ccccaccgga gggaaacgca 3420

gcgccggtgt cgctgtctgc ggaggggagt tacttggtga cgggtgggct gggcgccctt 3480

ggcctcctcg ttgcgcggtg gttggtggag cgcggggcgg ggcacctcgt gctgatcagc 3540

cggcacggat tgcccgaccg cgaggaatgg ggccgagatc agccgccaga ggtgcgcgcg 3600

cgcattgcgg cgatcgaggc gctggaggcg cagggcgcgc gggtcaccgt ggcggcggtc 3660

gacgtggccg atgccgaagg catggcggcg ctcttggcgg ccgtcgagcc gccgctgcgg 3720

ggggtcgtgc acgccgcggg tctgctcgac gacgggctgc tggcccacca ggacgctggt 3780

cggctcgccc gggtgttgcg ccccaaggtg gagggggcat gggtgctgca cacccttacc 3840

cgcgagcagc cgctggacct cttcgtactg ttttcctcgg cgtcgggcgt cttcggctcg 3900

atcggccagg gcagctacgc ggcaggcaat gcctttttgg acgcgctggc ggacctccgc 3960

cgaacgcagg ggctcgccgc cctgagcatc gcctggggcc tgtgggcgga gggggggatg 4020

ggctcgcagg cgcagcgccg ggaacacgag gcatcgggaa tctgggcgat gccgacgcgt 4080

cgtgccctgg cggcgatgga atggctgctc ggtacgcgcg cgacgcagcg cgtggtcatc 4140

cagatggatt gggcccatgc gggagcggct ccgcgcgacg cgagccgagg ccgcttctgg 4200

gatcggctgg taactgccac gaaagcgacc tcctcctcgg ccgtgccagc tgtggagcgc 4260

tggcgtaacg cgtctgtcgt ggagacccgc tcggcgctct acgagcttgt gcgcggcgtg 4320

gtcgccgggg tgatgggctt taccgatcag ggcacgctcg acgtgcgacg aggcttcgcc 4380

gagcagggcc tcgactccct gatggccgtg gaaatccgca aacggcttca gggtgagctg 4440

ggtatgccgc tgtcggcgac gctggcgttc gaccatccga ccgtggagcg gctggtggaa 4500

tacttgctga gccaggcgct ggagctgcag gaccgcaccg acgtgcgaag cgctcggttg 4560

ccggcgacag aggacccgat cgccatcgtg ggtgccgcct gccgcttccc gggcggggtc 4620

gaggacctgg agtcctactg gcagctgttg accgagggcg tggtggtcag caccgaggtg 4680

ccggccgacc ggtggaatgg ggcagacggg cgcggccccg gctcgggaga ggctcagaga 4740

cagacctacg tgcccagggg tggctttctg cgcgaggtgg agacgttcga tgcggcgttc 4800

ttccacatct cgcctcggga ggcgatgagc ctggacccgc aacagcggct gctgctggaa 4860

gtgagctggg aggcgatcga gcgcgcgggc caggacccgt cggcgctgcg cgagagcccc 4920

acgggcgtgt tcgtgggcgc gggccccaac gaatatgccg agcgggtgca ggacctcgcc 4980

gatgaggcgg cggggctcta cagcggcacc ggcaacatgc tcagcgttgc ggcgggacgg 5040

ctgtcatttt tcctgggcct gcacgggccg accctggctg tggatacggc gtgctcctcg 5100

tcgctcgtgg cgctgcacct cggctgccag agcttgcgac ggggcgagtg cgaccaagcc 5160

ctggttggcg gcgtcaacat gctgctctcg ccgaagacct tcgcgctgct ctcacggatg 5220

cacgcgcttt cgcccggcgg gcggtgcaag acgttctcgg ccgacgcgga cggctacgcg 5280

cgggccgagg gctgcgccgt ggtggtgctc aagcggctct ccgacgcgca gcgcgaccgc 5340

gaacccatcg tggcggtgat ccggggtacg gcgatcaatc atgatggccc gagcagcggg 5400

ctgacagtgc ccagcggccc tgcccaggag gcgctgttac gccaggcgct ggcgcacgca 5460

ggggtggttc cggccgacgt cgatttcgtg gaatgccacg ggaccgggac ggcgctgggc 5520

gacccgatcg aggtgcgggc gctgagcgac gtgtacgggc aagcccgccc tgcggaccga 5580

ccgctgatcc tgggagccgc caaggccaac cttgggcaca tggagcccgc ggcgggcctg 5640

gccggcttgc tcaaggcggt gctcgcgctg gggcaagagc aaataccagc ccagccggag 5700

ctgggcgagc tcaacccgct cttgccgtgg gaggcgctgc cggtggcggt ggcccgcgca 5760

gcggtgccgt ggccgcgcac ggaccggccg cgcttcgcgg gggtgagctc gttcgggatg 5820

agcggaacga acgcgcacgt ggtgctggaa gaggcgccgg cggtggagct ggggcctgcc 5880

gcgccggagc gctcggcgga gcttctggtg ctgtcgggca agagcgaggg ggcgctcgac 5940

gcgcaggcgg cgcggctgcg cgagcacctg gacatgcacc cggagctcgg gctcggggac 6000

gtggcgttca gcctggcgac gacgcgcagc gcgatgaacc accggctcgc ggtggcggtg 6060

acgtcgcgcg aggggctgct ggcggcgctc tcggccgtgg cgcaggggca gacgccgccg 6120

ggggcggcgc gctgcatcgc gagctcgtcg cgcggcaagc tggcgttgct gttcaccgga 6180

cagggcgcgc agacgccggg catgggccgg gggctttgcg cggcgtggcc agcgttccgg 6240

gaggcgttcg accggtgcgt ggcgctgttc gaccgggagc tggaccgccc gctgcgcgag 6300

gtgatgtggg cggagccggg gagcgccgag tcgttgctgc tcgaccagac ggcgttcacc 6360

cagcccgcgc tcttcacggt ggagtacgcg ctgacggcgc tgtggcggtc gtggggcgta 6420

gagccggagc tggtggctgg gcatagcgcc ggggagctgg tggcggcgtg cgtggcgggg 6480

gtgttctcgc tggaagatgg ggtgaggctc gtggcggcgc gcgggcggct gatgcagggg 6540

ctctcggcgg gcggcgcgat ggtgtcgctc ggagcgccgg aggcggaggt ggccgcggcg 6600

gtggcgccgc acgcggcgtc ggtgtcgatc gcggcggtca atgggccgga gcaggtggtg 6660

atagcgggcg tggagcgagc ggtgcaggcg atcgcggcgg ggttcgcggc gcgcggcgtg 6720

cgcaccaagc ggctgcatgt ctcgcacgcg tcccactcgc cgctgatgga accgatgctg 6780

gaggagttcg ggcgggtggc ggcgtcggtg acgtaccggc ggccaagcgt ttcgctggtg 6840

agcaacctga gcgggaaggt ggtcacggac gagctgagcg cgccgggcta ctgggtgcgg 6900

cacgtgcggg aggcggtgcg cttcgcggac ggggtgaagg cgctgcacga agccggcgcg 6960

gggacgttcc tcgaagtggg cccgaagccg acgctgctcg ggctgttgcc agcctgcctg 7020

ccggagacgg agccgacgct gctggcgtcg ttgcgcgccg ggcgcgagga ggctgcgggg 7080

gtgctcgagg cgctgggcag gctgtgggcc ggcggcggct cggtcagctg gccgggcgtc 7140

ttccccacgg ctgggcggcg ggtgccgctg ccgacctatc cgtggcagcg gcagcggtac 7200

tggcccgaca tcgagcctga cagccgtcgc cacgcagccg cggatccgac ccaaggctgg 7260

ttctatcgcg tggactggcc ggagatacct cgcagcctcc agaaatcaga ggaggcgagc 7320

cgcgggagct ggctggtatt ggcggataag ggtggagtcg gcgaggcggt cgctgcagcg 7380

ctgtcgacac gtggacttcc atgcgtcgtg atccatgcgc cggcagagac atccgcgacc 7440

gccgagctgg tgaccgaggc tgccggcggt cgaagcgatt ggcaggtagt gctctacctg 7500

tggggtctgg acgccgtcgt cggtgcggag gcgtcgatcg atgagatcgg cgacgcgacc 7560

cgtcgtgcta ccgcgccggt gctcggcttg gctcggtttc tgagcaccgt gtcttgttcg 7620

ccccgactct gggtcgtgac ccggggggca tgcatcgttg gcgacgagcc tgcgatcgcc 7680

ccttgtcagg cggcgttatg gggcatgggc cgggtggcgg cgctcgagca tcccggggcc 7740

tggggcgggc tcgtggacct ggatccccga gcgagcccgc cccaagccag cccgatcgac 7800

ggcgagatgc tcgtcaccga gctattgtcg caggagaccg aggatcagct cgccttccgc 7860

catgggcgcc ggcacgcggc acggctggtg gctgccccgc cacaggggca agcggcaccg 7920

gtgtcgctgt ctgcggaggc gagctacctg gtgacgggag gcctcggtgg gctgggcctg 7980

atcgtggccc agtggctggt ggagctggga gcgcggcact tggtgctgac cagccggcgc 8040

gggttgcccg accggcaggc gtggcgcgag cagcagccgc ctgagatccg cgcgcggatc 8100

gcagcggtcg aggcgctgga ggcgcggggt gcacgggtga ccgtggcagc ggtggacgtg 8160

gccgacgtcg aaccgatgac agcgctggtt tcgtcggtcg agcccccgct gcgaggggtg 8220

gtgcacgccg ctggcgtcag cgtcatgcgt ccactggcgg agacggacga gaccctgctc 8280

gagtcggtgc tccgtcccaa ggtggccggg agctggctgc tgcaccggct gctgcacggc 8340

cggccgctcg acctgttcgt gctgttctcg tcgggcgcag cggtgtgggg tagccatagc 8400

cagggtgcgt acgcggcggc caacgctttc ctcgacgggc tcgcgcatct tcggcgttcg 8460

caatcgctgc ctgcgttgag cgtcgcgtgg ggtctgtggg ccgagggagg catggcggac 8520

gcggaggctc atgcacgtct gagcgacatc ggggttctgc ccatgtcgac gtcggcagcg 8580

ttgtcggcgc tccagcgcct ggtggagacc ggcgcggctc agcgcacggt gacccggatg 8640

gactgggcgc gcttcgcgcc ggtgtacacc gctcgagggc gtcgcaacct gctttcggcg 8700

ctggtcgcag ggcgcgacat catcgcgcct tcccctccgg cggcagcaac ccggaactgg 8760

cgtggcctgt ccgttgcgga agcccgcgtg gctctgcacg agatcgtcca tggggccgtc 8820

gctcgggtgc tgggcttcct cgacccgagc gcgctcgatc ctgggatggg gttcaatgag 8880

cagggcctcg actcgttgat ggcggtggag atccgcaacc tccttcaggc tgagctggac 8940

gtgcggcttt cgacgacgct ggcctttgat catccgacgg tacagcggct ggtggagcat 9000

ctgctcgtcg atgtactgaa gctggaggat cgcagcgaca cccagcatgt ttggtcgttg 9060

gcgtcagacg agcccatcgc catcgtggga gccgcctgcc gcttcccggg cggggtggag 9120

gacctggagt cctactggca gctgttggcc gagggcgtgg tggtcagcgc cgaggtgccg 9180

gccgaccggt gggatgcggc ggactggtac gaccctgatc cggagatccc aggccggact 9240

tacgtgacca aaggcgcctt cctgcgcgat ttgcagagat tggatgcgac cttcttccgc 9300

atctcgcctc gcgaggcgat gagcctcgac ccgcagcagc ggttgctcct ggaggtaagc 9360

tgggaggcgc tcgagagcgc gggtatcgct ccggatacgc tgcgagatag ccccaccggg 9420

gtgttcgtgg gtgcggggcc caatgagtac tacacgcagc ggctgcgagg cttcaccgac 9480

ggagcggcag ggctgtacgg cggcaccggg aacatgctca gcgttacggc tggacggctg 9540

tcgtttttcc tgggtctgca cggcccgacg ctggccatgg atacggcgtg ctcgtcatcc 9600

ctggtcgcgc tgcacctcgc ctgccagagc ctgcgactgg gcgagtgcga tcaagcgctg 9660

gttggcgggg tcaacgtgct gctcgcgccg gagaccttcg tgctgctctc acggatgcgc 9720

gcgctttcgc ccgacgggcg gtgcaagacg ttctcggccg acgcggacgg ctacgcgcgg 9780

ggcgaggggt gcgccgtggt ggtgctcaag cggctgcgcg atgcgcagcg cgccggcgac 9840

tccatcctgg cgctgatccg gggaagcgcg gtgaaccacg acggcccgag cagcgggctg 9900

accgtgccca acggaccggc ccagcaagca ttgctgcgcc aggcgctttc gcaagcaggc 9960

gtgtctccgg tcgacgttga ttttgtggag tgtcacggga cagggacggc gctgggcgac 10020

ccgatcgagg tgcaggcgct gagcgaggtg tatggtccag ggcgctccgg ggatcgaccg 10080

ctggtgctgg gggccgtcaa ggccaacgtc gcgcatctgg aggcggcatc cggcttggcc 10140

agcctgctca aggccgtgct tgcgctgcgg cacgagcaga tcccggccca gccggagctg 10200

ggcgagctca acccgcactt gccgtggaac acgctgccgg tggcggtgcc acgtaatgcg 10260

gtgccgtggg ggcgcggcgc acgcccgcgt cgggccggcg tgagcgcgtt cgggttgagc 10320

ggaaccaacg tgcatgtcgt gctggaggag gcaccggagg tggagccggc gcccgcggcg 10380

ccggcgcgac cggtggagct ggtcgtgcta tcggccaaga gcgcggcggc gctggacgcc 10440

gcggcggaac ggctctcggc gcacctgtcc gcgcacccgg agctgagcct cggcgacgtg 10500

gcgttcagcc tggcgacgac gcgcagcccg atggagcacc ggctcgccat cgcgacgacc 10560

tcgcgcgagg ccctgcgagg cgcgctggac gccgcggcgc agcaaaagac gccgcagggc 10620

gcggtgcgcg gcaaggccgt gtcctcacgc ggtaagctgg ctttcctgtt caccggacag 10680

ggcgcgcaaa tgccgggcat gggccgtggg ctgtacgaaa cgtggcctgc gttccgggag 10740

gcgttcgacc ggtgcgtggc gctcttcgat cgggagatcg accagcctct gcgcgaggtg 10800

atgtgggctg cgccgggcct cgctcaggcg gcgcggctcg atcagaccgc gtacgcgcag 10860

ccggctctct ttgcgctgga gtacgcgctg gctgccctgt ggcgttcgtg gggcgtggag 10920

ccgcacgtac tgctcggtca tagcatcggc gagctggtcg ccgcctgcgt ggcgggcgtg 10980

ttctcgctcg aagatgcggt gaggttggtg gccgcgcgcg ggcggctgat gcaggcgcta 11040

cccgccggcg gtgccatggt agccatcgca gcgtccgagg ccgaggtggc cgcctccgtg 11100

gcgccccacg ccgccacggt gtcgatcgcc gcggtcaacg gtcctgacgc cgtcgtgatc 11160

gccggcgccg aggtacaggt gctcgccctc ggcgcgacgt tcgcggcgcg tgggatacgc 11220

acgaagaggc tcgccgtctc ccatgcgttc cactcgccgc tcatggatcc gatgctggaa 11280

gacttccagc gggtcgctgc gacgatcgcg taccgcgcgc cggaccgccc ggtggtgtcg 11340

aatgtcaccg gccacgtcgc aggccccgag atcgccacgc ccgagtattg ggtccggcat 11400

gtgcgaagcg ccgtgcgctt cggcgacggg gcaaaggcgt tgcatgccgc gggtgccgcc 11460

acgttcgtcg agattggccc gaagccggtc ctgctcgggc tgttgccagc gtgcctcggg 11520

gaagcggacg cggtcctcgt gccgtcgcta cgcgcggacc gctcggaatg cgaggtggtc 11580

ctcgcggcgc tcggggcttg gtatgcctgg gggggtgcgc tcgactggaa gggcgtgttc 11640

cccgatggcg cgcgccgcgt ggctctgccc atgtatccat ggcagcgtga gcgccattgg 11700

atggacctca ccccgcgaag cgccgcgcct gcagggatcg caggtcgctg gccgctggct 11760

ggtgtcgggc tctgcatgcc cggcgctgtg ttgcaccacg tgctctcgat cggaccacgc 11820

catcagcctt tcctcggtga tcacctcgtg tttggcaagg tggtggtgcc cggcgccttt 11880

catgtcgcgg tgatcctcag catcgccgcc gagcgctggc ccgagcgggc gatcgagctg 11940

acaggcgtgg agttcctgaa ggccatcgcg atggagcccg accaggaggt cgagctccac 12000

gccgtgctca cccccgaagc cgccggggat ggctacctgt tcgagctggc gaccctggcg 12060

gcgccggaga ccgaacgccg atggacgacc cacgcccgcg gtcgggtgca gccgacagac 12120

ggcgcgcccg gcgcgttgcc gcgcctcgag gtgctggagg accgcgcgat ccagcccctc 12180

gacttcgccg gattcctcga caggttatcg gcggtgcgga tcggctgggg gccgctttgg 12240

cgatggctgc aggacgggcg cgtcggcgac gaggcctcgc ttgccaccct cgtgccgacc 12300

tatccgaacg cccacgacgt ggcgcccttg cacccgatcc tgctggacaa cggctttgcg 12360

gtgagcctgc tggcaacccg gagcgagccg gaggacgacg ggacgccccc gctgccgttc 12420

gccgtggaac gggtgcggtg gtggcgggcg ccggttggaa gggtgcggtg tggcggcgtg 12480

ccgcggtcgc aggcattcgg tgtctcgagc ttcgtgctgg tcgacgaaac tggcgaggtg 12540

gtcgctgagg tggagggatt tgtttgccgc cgggcgccgc gagaggtgtt cctgcggcag 12600

gagtcgggcg cgtcgactgc agccttgtac cgcctcgact ggcccgaagc gcccttgccc 12660

gatgcgcctg cggaacggat ggaggagagc tgggtcgtgg tggcagcacc tggctcggag 12720

atggccgtgg cgctcgcaac acggctcaac cgctgcgtcc tcgccgaacc ccaaggcctc 12780

gagacggccc tcgcgggggt gtctcccgca ggtgtgatct gcctctggga acctggagcc 12840

cacgaggaag ctccggcggc ggcgcagcgt gtggcgaccg agggcctctc ggtggtgcag 12900

gcgctcaggg atcgcgcggt gcgcctctgg tgggtgacca cgggcgcagt ggctgtcgag 12960

gccggcgagc gggtgcaggt cgccacagcg gcggtgtggg gcctgggccg gacagtgatg 13020

caggagcgcc cggagctcag ctgcactctg gtggatttgg agccggaggc cgatgcagcg 13080

cgttcagctg acgttctgtt gcgggagctc ggtcgcgctg acgacgagac ccaggtggtt 13140

ttccgttccg gaaagcgccg cgtagggcgg ctggtcaaag cgacaacccc cgaagggctc 13200

ttggtccctg acgcagaatc ctatcgactg gaggctgggc agaagggcac attggaccag 13260

ctccgcctcg cgccggcaca gcgccgggca cctggcccgg gcgaggtcga gatcaaggta 13320

accgcctcgg ggctcaactt ccggaccgtc ctcgctgtgc tgggaatgta tccgggcgac 13380

gctgggccga tgggcggaga ttgtgccggt atcgtcacgg cggtgggcca gggggtgcac 13440

cacctctcgg tcggcgatgc tgtcatgacg ctggggacgt tgcatcgatt cgtcacggtc 13500

gacgcgcggc tggtggtccg gcagcctgca gggctgactc ccgcgcaggc agctacggtg 13560

ccggtcgcgt tcctgacggc ctggctcgct ctgcacgacc tggggaatct gcagcgcggc 13620

gagcgggtgc tgatccatgc tgcggccggc ggtgtgggca tggccgcggt gcaaatcgcc 13680

cgatggatag gggccgaggt gttcgccacg gcgagcccgt ccaagtgggc agcgcttcag 13740

gccatgggcg tgccgcgcac gcacatcgcc agctcgcgga cgctggagtt tgctgagacg 13800

ttccggcagg tcaccggcgg ccggggcgtg gacgtggtgc tcaacgcgct ggccggcgag 13860

ttcgtggacg cgagcctgtc cctgctgtcg acgagcgggc ggttcctcga gatgggcaag 13920

accgacatac gggatcgagc cgcggtcgcg gcggcgcatc ccggtgttcg ctatcgggta 13980

ttcgacatcc tggagctcgc tccggatcga actcgagaga tcctcgagcg cgtggtcgag 14040

ggctttgctg cgggacatct gcgcgcattg ccggtgcatg cgttcgcgat caccaaggcc 14100

gaggcagcgt ttcggttcat ggcgcaagcg cggcatcagg gcaaggtcgt gctgctgccg 14160

gcgccctccg ccgcgccctt ggcgccgacg ggcaccgtac tgctgaccgg tgggctggga 14220

gcgttggggc tccacgtggc ccgctggctc gcccagcagg gcgtgccgca catggtgctc 14280

acaggtcggc ggggcctgga tacgccgggc gctgctaaag ccgtcgcgga gatcgaagcg 14340

ctcggcgctc gggtgacgat cgcggcgtcg gatgtcgccg atcggaatgc gctggaggct 14400

gtgctccagg ccattccggc ggagtggccg ttacagggcg tgatccatgc agccggagcg 14460

ctcgatgatg gtgtgcttga tgagcagacc accgaccgct tctcgcgggt gctggcaccg 14520

aaggtgactg gcgcctggaa tctgcatgag ctcacggcgg gcaacgatct cgctttcttc 14580

gtgctgttct cctccatgtc ggggctcttg ggctcggccg ggcagtccaa ctatgcggcg 14640

gccaacacct tcctcgacgc gctggccgcg catcggcggg ccgaaggcct ggcggcgcag 14700

agcctcgcgt ggggcccatg gtcggacgga ggcatggcag cggggctcag cgcggcgctg 14760

caggcgcggc tcgctcggca tgggatggga gcgctgtcgc ccgctcaggg caccgcgctg 14820

ctcgggcagg cgctggctcg gccggaaacg cagctcgggg cgatgtcgct cgacgtgcgt 14880

gcggcaagcc aagcttcggg agcggcagtg ccgcctgtgt ggcgcgcgct ggtgcgcgcg 14940

gaggcgcgcc atgcggcggc tggggcgcag ggggcattgg ccgcgcgcgt tggggcgctg 15000

cccgaggcgc gtcgcgccga cgaggtgcgc aaggtcgtgc aggccgagat cgcgcgcgtg 15060

ctttcatgga gcgccgcgag cgccgtgccc gtcgatcggc cgctgtcgga cttgggcctc 15120

gactcgctca cggcggtgga gctgcgcaac gtgctcggcc agcgggtggg tgcgacgctg 15180

ccggcgacgc tggcattcga tcacccgacg gtcgacgcgc tcacgcgctg gctgctcgat 15240

aaggtcctgg tcgtggccga gccgagcgta tcgcccgcaa agtcgtcgcc gcaggtcgcc 15300

ctcgacgagc ccattgcggt gatcggcatc ggctgccgtt tcccaggcgg cgtgaccgat 15360

ccggagtcgt tttggcggct gctcgaagag ggcagcgatg ccgtcgtcga ggtgccgcat 15420

gagcgatggg acatcgacgc gttctatgat ccggatccgg atgtgcgcgg caagatgaca 15480

acacgctttg gcggcttcct gtccgatatc gaccggttcg agccggcctt cttcggcatc 15540

tcgccgcgcg aagcgacgac gatggatccg cagcagcggc tgctcctgga gacgagctgg 15600

gaggcgttcg agcgcgccgg gattttgccc gagcggctga tgggcagcga taccggcgtg 15660

ttcgtggggc tcttctacca ggagtacgct gcgctcgccg gcggcatcga ggcgttcgat 15720

ggctatctag gcaccggcac cacggccagc gtcgcctcgg gcaggatctc ttatgtgctc 15780

gggctaaagg ggccgagcct gacggtggac accgcgtgct cctcgtcgct ggtcgcggtg 15840

cacctggcct gccaggcgct gcggcggggc gagtgttcgg tggcgctggc cggcggcgtg 15900

gcgctgatgc tcacgccggc gacgttcgtg gagttcagcc ggctgcgagg cctggctccc 15960

gacggacggt gcaagagctt ctcggccgca gccgacggcg tggggtggag cgaaggctgc 16020

gccatgctcc tgctcaaacc gcttcgcgat gcgcagcgcg atggggatcc gatcctggcg 16080

gtgatccgcg gcaccgcggt gaaccaggat gggcgcagca acgggctgac ggcgcccaac 16140

ggatcgtcgc agcaagaggt gatccgtcgt gccctggagc aggcggggct ggctccggcg 16200

gacgtcagct acgtcgagtg ccacggcacc ggcacgacgt tgggggaccc catcgaagtg 16260

caggccctgg gcgccgtgct ggcacagggg cgaccctcgg accggccgct cgtgatcggg 16320

tcggtgaagt ccaatatcgg acatacgcag gctgcggcgg gcgtggccgg tgtcatcaag 16380

gtggcgctgg cgctcgagcg cgggcttatc ccgaggagcc tgcatttcga cgcgcccaat 16440

ccgcacattc cgtggtcgga gctcgccgtg caggtggccg ccaaacccgt cgaatggacg 16500

agaaacggcg tgccgcgacg agccggggtg agctcgtttg gcgtcagcgg gaccaacgcg 16560

cacgtggtgc tggaggaggc gccagcggcg gcgttcgcgc ccgcggcggc gcgttcagcg 16620

gagcttttcg tgctgtcggc gaagagcgcc gcggcgctgg acgcgcaggc ggcgcggctt 16680

tcggcgcatg tcgttgcgca cccggagctc ggcctcggcg acctggcgtt cagcctggcg 16740

acgacccgca gcccgatgac gtaccggctc gcggtggcgg cgacctcgcg cgaggcgctg 16800

tctgcggcgc tcgacacagc ggcgcagggg caggcgccgc ccgcagcggc tcgcggccac 16860

gcttccacag gcagcgcccc aaaggtggtt ttcgtctttc ctggccaggg ctcccagtgg 16920

ctgggcatgg gccaaaagct cctctcggag gagcccgtct tccgcgacgc gctctcggcg 16980

tgtgaccgag cgattcaggc cgaagccggc tggtcgctgc tcgccgagct cgcggccgat 17040

gagaccacct cgcagctcgg ccgcatcgac gtggtgcagc cggcgctgtt cgcgatcgag 17100

gtcgcgctgt cggcgctgtg gcggtcgtgg ggcgtcgagc cggatgcagt ggtaggccac 17160

agcatgggcg aagtggcggc cgcgcacgtc gccggcgccc tgtcgctcga ggatgctgta 17220

gcgatcatct gccggcgcag cctgctgctg cggcggatca gcggccaagg cgagatggcg 17280

gtcgtcgagc tttccctggc cgaggccgag gcagcgctcc tgggctacga agaccggctc 17340

agcgtggcgg tgagcaacag cccgcgatcg acggtgctgg cgggcgagcc ggcagcgctc 17400

gcagaggtgc tggcgatcct tgccgcaaag ggggtgttct gccgtcgagt caaggtggac 17460

gtcgccagcc acagcccaca gatcgacccg ctgcgcgacg agctattggc agcattgggc 17520

gagctcgagc cgcgacaagc gaccgtgacg atgcgctcga cggtgacgag cacgatcgtg 17580

gcgggcccgg agctcgtggc gagctactgg gcggacaacg ttcgacagcc ggtgcgcttc 17640

gccgaagcgg tgcaatcgtt gatggaaggc ggtcatgggc tgttcgtgga gatgagcccg 17700

catccgatcc tgacgacatc ggtcgaggag atccgacggg cgacgaagcg ggagggagtc 17760

gcggtggggt cgttgcggcg tggacaggac gagcgcctgt ccatgttgga ggcgctggga 17820

gcgctctggg tacacggcca ggcggtgggc tgggagcggc tgttctccgc gggcggcgcg 17880

ggcctccgtc gcgtgccgct gccgacctat ccctggcagc gcgagcggta ctgggtcgat 17940

gcgccgaccg gcggcgcggc gagcggcagc cgctttgctc atgcgggcag tcacccgctc 18000

ctgggtgaaa tgcagaccct gtcgacccag aggagcacgc gcgtgtggga gacgacgctg 18060

gatctcaaac ggctgccgtg gctcggcgat caccgggtgc agggggcggt cgtgttcccg 18120

ggcgcggcgt acctggagat ggcgctttcg tccggggccg aggccttggg tgacggtccg 18180

ctccaggtca gcgatgtggt gctcgccgag gcgctggcct tcgcggatga tacgccggcg 18240

gcggtgcagg tcatggcgac cgaggagcga ccaggccgcc tgcaattcca cgttgcgagc 18300

cgggtgccgg gccacggcgg tgctgccttt cgaagccatg cccgcggggt gctgcgccag 18360

atcgagcgcg ccgaggtccc ggcgaggctg gatctggccg cgcttcgtgc ccggcttcag 18420

gccagcgcac ccgctgcggc tacctatgcg gcgctggccg agatggggct cgagtacggc 18480

ccagcgttcc aggggcttgt cgagctgtgg cggggggagg gcgaggcgct gggacgtgtg 18540

cggctccccg aggccgccgg ctccccagcc gcgtgccggc tccaccccgc gctcttggat 18600

gcgtgcttcc acgtgagcag cgccttcgct gaccgcggcg aggcgacgcc atgggtaccc 18660

gtggaaatcg gctcgctgcg gtggttccag cggccgtcgg gggagctgtg gtgtcatgcg 18720

cggagtgtga gccacggaaa gccaacaccc gaccggcgga gtaccgactt ctgggtggtc 18780

gacagcacgg gcgcgatcgt cgccgagatc tccgggctcg tggcgcagcg gctcgcggga 18840

ggtgtacgcc ggcgcgaaga agacgactgg ttcatggagc cggcttggga accgaccgcg 18900

gtccccggat ccgaggtcat ggcgggccgg tggctgctca tcggctcggg cggcgggctc 18960

ggcgctgcgc tcgactcggc gctgacggaa gctggccatt ccgtcgtcca cgcgacaggg 19020

cacggcacga gcgccgccgg gttgcaggcg ctcttgacgg cgtccttcga cggccaggcc 19080

ccgacgtcgg tggtgcacct cggcagcctc gatgagcgtg gcgggctcga cgcggacgcg 19140

cccttcgacg ccgatgcgct cgaggagtcg ctggtgcgcg gctgcgacag cgtgctctgg 19200

accgtgcagg ccgtggccgg ggcgggcttc cgagatcctc cgcggttgtg gctcgtgaca 19260

cgcggcgctc aggccatcgg cgccggcgac gtctctgtgg cgcaagcgcc gctcctgggg 19320

ctgggccgcg ttatcgcctt ggagcacgcc gagctgcgct gcgctcggat cgacctcgat 19380

ccagcgcggc gcgacggaga agtcgatgcg ctgcttgccg agctgttggc cgacgacgcc 19440

gaggaggaag tcgcgtttcg cggcggtgag cggcgcgtgg cccggctcgt ccgaaggccg 19500

cccgagaccg actgccgaga gaaaatcgag cccgcggaag gccggccgtt ccggctggag 19560

atcgatgggt ccggcgtgct cgacgacctg gtgctccgag ccacggagcg gcgccctcct 19620

ggcccgggcg aggtcgagat cgccgtcgag gcggcggggc tcaactttct cgacgtgatg 19680

agggccatgg ggatctaccc tgggcctggg gacggtccgg ttgcgctggg cgccgagtgc 19740

tccggccgaa ttgtcgcgat gggcgaaggt gtcgagagcc ttcgtatcgg ccaggacgtc 19800

gtggccgtcg cgcccttcag tttcggcacc cacgtcaccg tcgacgcccg gatggtcgca 19860

cctcgccccg cggcgctgac ggccgcgcag gcagccgcgc tgcccgtcgc attcatgacg 19920

gcctggtacg gtctcgtcca tctggggagg ctccgggccg gcgagcgcgt gctcatccac 19980

tcggcgacgg ggggcaccgg ccttgctgct gtgcagatcg cccgccacct cggcgcggag 20040

atatttgcga ccgctggtac accggagaaa cgggcgtggc tgcgcgagca ggggatcgcg 20100

cacgtgatgg actcgcgctc gctggacttc gccgagcaag tgctggccgc gacgaagggc 20160

gagggggtcg acgtcgtgtt gaactcgctg tctggcgccg cgatcgacgc gagcctttcg 20220

accctcgtgc cggacggccg cttcatcgag ctcggcaaga cggacatcta tgcagatcgc 20280

tcgctggggc tcgctcactt caggaagagc ctgtcctaca gcgccgtcga tcttgcgggt 20340

ttggccgtgc gtcggcccga gcgcgtcgca gcgctgctgg cggaggtggt ggacctgctc 20400

gcacggggag cgctgcagcc gcttccggta gagatcttcc ccctctcgcg ggccgcggac 20460

gcgttccgga aaatggcgca agcgcagcat ctcgggaagc tcgtgctcgc gctggaggac 20520

ccggacgtgc ggatccgcgt ttcgggcgaa tccggcgtcg ccatccgcgc ggacggcacc 20580

tacctcgtga ccggcggtct gggtgggctc ggtctgagcg tggctggatg gctggccgag 20640

cagggggctg ggcatctggt gctggtgggc cgctccggtg cggtgagcgc ggagcagcag 20700

acggctgtcg ccgcgctcga ggcgcacggc gcgcgtgtga cggtagcgag ggcagacgtc 20760

gccgatcggg cgcagatcga gcgcatcctc cgcgaggtta ccgcgtcggg gatgccgctc 20820

cgcggcgtcg ttcatgcggc cggtatcctg gacgacgggc tgctgatgca gcaaaccccc 20880

gcgcggttcc gcgcggtcat ggcgcccaag gtccgagggg ccttgcacct gcatgcgttg 20940

acacgcgaag cgccgctctc cttcttcgtg ctgtacgctt cgggagcagg gctcttgggc 21000

tcgccgggcc agggcaatta cgccgcggcc aacacgttcc tcgacgctct ggcgcaccac 21060

cggagggcgc aggggctgcc agcgttgagc gtcgactggg gcctgttcgc ggacgtgggt 21120

ctggccgccg ggcagcaaaa tcgcggcgcg cggctggtca cccgcgggac gcggagcctc 21180

acccccgacg aagggctgtg ggcgctcgag cgtctgctcg acggcgatcg cacccaggcc 21240

ggggtcatgc cgttcgacgt gcggcagtgg gtggagttct acccggcggc ggcatcttcg 21300

cggaggttgt cgcggctggt gacggcacgg cgcgtggctt ccggtcggct cgccggggat 21360

cgggacctgc tcgaacggct cgccaccgcc gaggcgggcg cgcgggcagg aatgctgcag 21420

gaggtcgtgc gcgcgcaggt ctcgcaggtg ctgcgcctcc ccgaaggcaa gctcgacgtg 21480

gatgcgccgc tcacgagcct gggaatggac tcgctgatgg ggctagagct gcgcaaccgc 21540

atcgaggccg tgctcggcat caccatgccg gcgaccctgc tgtggaccta ccccacggtg 21600

gcagcgctga gtgcgcatct ggctagccat gtcgtctcta cgggggatgg ggaatccgtg 21660

cgcccgcctg atacagggag cgtggctcca atgacccacg aagtcgcttc gctcgacgaa 21720

gacgggttgt tcgcgttgat tgatgagtca ctcgcgcgtg cgggaaagag gtga 21774

<210> 5

<211> 11433

<212> DNA

<213> Sorangium cellulosum

<400> 5

atgagtcact cgcgcgtgcg ggaaagaggt gattgcgtga cagaccgaga aggccagctc 60

ctggagcgct tgcgtgaggt tactctggcc cttcgcaaga cgctgaacga gcgcgatacc 120

ctggagctcg agaagaccga gccgatcgcc atcgtgggga tcggctgccg cttccccggc 180

ggagcgggca ctccggaggc gttctgggag ctgctcgacg acgggcgcga cgcgatccgg 240

ccgctcgagg agcgctgggc gctcgtaggt gtcgacccag gcgacgacgt accgcgctgg 300

gcggggctgc tcaccgaggc catcgacggc ttcgacgccg cgttcttcgg tatcgccccc 360

cgggaggcac ggtcgctcga cccgcagcat cgcctgctgc tggaggtcgc ctgggagggg 420

ttcgaagacg ccggcatccc gcccaggtcc ctcgtcggga gccgcaccgg cgtgttcgtc 480

ggcgtctgcg ccacggagta cctccacgcc gccgtcgcgc accagccgcg cgaagagcgg 540

gacgcgtaca gcaccaccgg caacatgctc agcatcgccg ccggacggct atcgtacacg 600

ctggggctgc agggaccttg cctgaccgtc gacaccgcgt gctcgtcatc gctggtggcc 660

attcacctcg cctgccgcag cctgcgcgct cgagagagcg atctcgcgct ggcgggaggg 720

gtcaacatgc tcctctcccc cgacacgatg cgagctctgg cgcgcaccca ggcgctgtcg 780

cccaatggcc gttgccagac cttcgacgcg tcggccaacg ggttcgtccg tggggagggc 840

tgcggtctga tcgtgctcaa gcgattgagc gacgcgcggc gggatgggga ccggatctgg 900

gcgctgatcc gaggatcggc catcaatcag gacggccggt cgacggggtt gacggcgccc 960

aacgtgctcg cccagggggc gctcttgcgc gaggcgctgc ggaacgccgg cgtcgaggcc 1020

gaggccatcg gttacatcga gacccacggg gcggcgacct cgctgggcga ccccatcgag 1080

atcgaagcgc tgcgcgccgt ggtggggccg gcgcgagccg acggagcgcg ctgcgtgctg 1140

ggcgcggtga agaccaacct cggccacctg gagggcgctg ccggcgtggc gggcctgatc 1200

aaggcgacgc tttcgctaca tcacgagcgc atcccgagga acctcaactt tcgtacgctc 1260

aatccgcgga tccggatcga ggggaccgcg ctcgagttgg cgaccgagcc ggtgccctgg 1320

ccgcggacgg gccggacgcg cttcgcggga gtgagctcgt tcgggatgag cgggaccaac 1380

gcgcatgtgg tgttggagga ggcgccggcg gtggagcctg aggccgcggc ccccgagcgc 1440

gctgcggagc tgttcgtcct gtcggcgaag agcgtggcgg cgctggatgc gcaggcagcc 1500

cggctgcggg atcacctgga gaagcatgtc gagcttggcc tcggcgatgt ggcgttcagc 1560

ctgacgacga cgcgcagcgc gatggagcac cggctggcgg tggccgcgag ctcgcgcgag 1620

gcgctgcgag gggcgctttc ggccgcagcg caggggcaca cgccgccggg agctgtgcgt 1680

gggcgggcct cgggcggcag cgcgccgaag gtggtcttcg tgtttcccgg ccagggctcg 1740

cagtgggtgg gcatgggccg aaagctcatg gccgaagagc cggtcttccg ggcggcgctg 1800

gagggttgcg accgggccat cgaggcggaa gcgggctggt cgctgctcgg ggagctctcc 1860

gccgacgagg ccgcctcgca gctcgggcgc atcgacgtgg ttcagccggt gctcttcgcc 1920

atggaagtag cgctttctgc gctgtggcgg tcgtggggag tggagccgga agcggtggtg 1980

ggccacagca tgggcgaggt tgcggcggcg cacgtggccg gcgcgctgtc gctcgaggac 2040

gctgtggcga tcatctgccg gcgcagccgg ctgctgcggc ggatcagcgg tcagggggag 2100

atggcgctgg tcgagctgtc gctggaggag gccgaggcgg cgctgcgtgg ccatgagggt 2160

cggctgagcg tggcggtgag caacagcccg cgctcgaccg tgctcgcagg cgagccggcg 2220

gcgctctcgg aggtgctggc ggcgctgacg gccaaggggg tgttctggcg gcaggtgaag 2280

gtggacgtcg ccagccatag cccgcaggtc gacccgctgc gcgaagagct ggtcgcggcg 2340

ctgggagcga tccggccgcg agcggctgcg gtgccgatgc gctcgacggt gacgggcggg 2400

gtgattgcgg gtccggagct cggagcgagc tactgggcgg gcaatcttcg gcagccggtg 2460

cgcttcgctg cggcggcgcg agcgctgctg gaaggtggcc ccacgctgtt catcgagatg 2520

agcccgcacc cgatcctggt gccgcccctg gacgagatcc agacggcggt cgagcaaggg 2580

ggcgctgcgg tgggctcgct gcggcgaggg caggacgagc gcgcgacgct gctggaggcg 2640

ctggggacgc tgtgggcgtc cggctatccg gtgagctggg ctcggctgtt ccccgcgggc 2700

ggcaggcggg ttccgctgcc gacctatccc tggcagcacg agcggtgctg gatcgaggtc 2760

gagcctgaag cccgccgcct cgccgcagcc gaccccacca aggactggtt ctaccggacg 2820

gactggcccg aggtgccccg cgccgccccg aaatcggaga cagctcatgg gagctggctg 2880

ctgttggccg acaggggtgg ggtcggtgag gcggtcgctg cagcgctgtc gacgcgcgga 2940

ctttcctgca ccgtgcttca tgcgtcggct gacgcctcca ccgtcgccga gcaggtatcc 3000

gaagctgcca gtcgccgaaa cgactggcag ggagtcctct acctgtgggg cctcgacgcc 3060

gtcgtcgatg ctggggcatc ggccgacgac gtcagcgagg ctacccgccg tgccaccgca 3120

cccgtccttg ggctggttcg attcctgagc gctgcgcccc atcctcctcg cttctgggtg 3180

gtgacccgcg gggcatgcac ggtgggcggc gagccagagg tctctctttg ccaagcggcg 3240

ttgtggggcc tcgcgcgcgt cgtggcgctg gagcatcccg ctgcctgggg tggcctcgtg 3300

gacctggatc ctcagaagag cccgacggag atcgagcccc tggtggccga gctgctttcg 3360

ccggacgccg aggatcagct ggcgttccgt agcggtcgcc ggcacgcagc acgccttgta 3420

gccgccccgc cggagggcga cgtcgcaccg atatcgctgt ccgcggaggg aagctacctg 3480

gtgacgggcg ggctgggcgg ccttggtctg ctcgtggctc ggtggctggt ggagcgggga 3540

gctcgacatc tggtgctcac cagccggcac gggctgccag agcgacaggc gtcgggcgga 3600

gagcagccgc cggaggcccg cgcgcgcatc gcagcggtcg aggggctgga agcgcagggc 3660

gcgcgggtga ccgtggcagc ggtggatgtc gccgaggccg atcccatgac ggcgctgctg 3720

gccgccatcg agcccccgtt gcgcggggtg gtgcacgccg ccggcgtctt ccccgtgcgt 3780

cccctggcgg agacggacga ggccctgctg gagtcggtgc tccgtcccaa ggtggccggg 3840

agctggctgc tgcaccggct gctgcgcgac cggcctctcg acctgttcgt gctgttctcg 3900

tcgggcgcgg cggtgtgggg tggcaaaggc caaggcgcat acgccgcggc caatgcgttc 3960

ctcgacgggc tcgcgcacca tcgccgcgcg cgctcgctgc cggcgttgag cctcgcctgg 4020

ggcttatggg ccgagggagg catggttgat gcaaaggctc atgcacgtct gagcgacatc 4080

ggggtcctgc ccatggccac ggggccggcc ttgtcggcgc tggagcgcct ggtgaagacc 4140

agcgctgtcc agcgttcggt cacacggatg gactggacgc gcttcgcgcc cgtctatgcc 4200

gcgcgagggc ggcgcaactt gctttcggct ctggtcgcgg aggacgagcg cactgcgtct 4260

ccccctgtgc cgacggcaaa ccgaatctgg cgcggcctgt ccgttgcgga gagccgctca 4320

gccctctacg agctcgttcg cggcatcgcc gcccgggtgc tgggcttcgc cgacccgggc 4380

gcgctcgacg tcggccgagg cttcgccgag caggggctcg actccctgat ggctctggag 4440

atccgtaacc gccttcagcg cgagctgggc gaacggctgt cggcgactct ggccttcgac 4500

cacccgacgg tggagcggct ggtggcgcat ctcctcaccg acgtgctgaa gctggaggac 4560

cggagcgaca cccggcacat ccggtcggtg gcggcggatg acgacatcgc catcgtcggt 4620

gccgcctgcc ggttcccagg tggggatgaa agcctggaga catactggcg gcatctggcc 4680

gagggcatgg tggtcagcgc cgaggtgcca gccgaccggt ggcgcgcggc ggactggtac 4740

gaccccgatc cggaggttcc gggccggacc tatgtggcca agggtgcctt cctccgcgat 4800

gtgcgcagct tggatgcggc gttcttctcc atttcccctc gtgaggcgat gagcctggac 4860

ccgcaacagc ggctgttgct ggaggtgagc tgggaggcga tcgagcgcgc tggccaggac 4920

ccgatggcgc tgcgcgagac cgccacgggc gtgttcgtgg gcatgatcgg gagcgagcac 4980

gccgagcggg tgcagggcct cgacgacgac gcggcgttgc tgtacggcac caccggcaac 5040

ctgctcagcg tcgccgctgg acggctgtcg ttcttcctgg gtctgcacgg cccgacgatg 5100

acggtggaca ccgcctgctc gtcgtcgctg gtggcgttgc acctcgcctg ccagagcctg 5160

cgattgggcg agtgcgacca ggccctggcc ggcgggtcca gcgtgctttt gtcgccgcgg 5220

tcattcgtcg cggcgtcgcg catgcgtttg ctttcgccag atgggcggtg caagacgttc 5280

tcggccgctg cagacggctt tgcgcgggcc gagggctgcg ccgtggtggt gctgaagcag 5340

ctccgtgacg cgcagcgcga ccgcgacccc atcctggcgg tggtcaggag cacggcgatc 5400

aaccacgatg gcccgagcag cgggctcacg gtgcccagcg gtcctgccca gcaggcgttg 5460

ctaggccagg cgctggcgca agcgggcgtg gcgccggccg aggtcgattt cgtggagtgc 5520

cacgggacgg ggacagcgct gggtgacccg atcgaggtgc aggcgctggg cgcggtgtat 5580

gggcggggcc gccccgcgga gcggccgctc tggctgggcg ctgtcaaggc caacctcggg 5640

cacctggagg ccgcggcggg cttggccggc gtgctcaagg tgctcttggc gctggagcac 5700

gagcagattc cggctcaacc ggagctcgac gagctcaacc cgcacatccc gtgggcagag 5760

ctgccagtgg ccgttgtccg cagggcggtc ccctggccgc gcggcgcgcg cccgcgtcgt 5820

gcaggcgtga gcgctttcgg cctgagcggg accaacgcgc atgtggtgtt ggaggaggcg 5880

ccgacggtgg agcctggggc cgcggccccc gagcgcgcag cggagctgtt cgtcctgtcg 5940

gcgaagagcg tggcggcgct ggatgcgcag gcagcccggc tgcgggatca cctggagaag 6000

catgtcgagc ttggcctcgg cgatgtggcg ttcagcctgg cgacgacgcg cagcgcgatg 6060

gagcaccggc tggcggtggc cgcgagctcg cgcgaggcgc tgcgaggggc gctttcggcc 6120

gcagcgcagg ggcacacgcc gccgggagct gtgcgtgggc gggcctcggg cggcagcgcg 6180

ccgaaggtgg tcttcgtgtt tcccggccag ggctcgcagt gggtgggcat gggccgaaag 6240

ctcatggccg aagagccggt cttccgggcg gcgctggagg gttgcgaccg ggccatcgag 6300

gcggaagcgg gctggtcgct gctcggggag ctctccgccg acgaggccgc ctcgcagctc 6360

gagcgcatcg acgtggttca gccggtgctc ttcgccatgg aagtagcgct ttctgcgctg 6420

tggcggtcgt ggggagtgga gccggaagcg gtggtgggcc acagcatggg cgaggttgcg 6480

gcggcgcacg tggccggcgc gctgtcgctc gaggacgctg tggcgatcat ctgccggcgc 6540

agccggctgc tgcggcggat cagcggccag ggggagatgg cgctggtcga gctgacgctg 6600

gaggaggccg aggcggcgct gcgtggccat gagggtcggc tgagcgtggc ggtgagcaac 6660

agcccgcgct cgaccgtgct cgcaggcgag ccggcggcgc tctcggaggt gctggcggcg 6720

ctgacggcca agggggtgtt ctggcggcag gtgaaggtgg acgtcgccag ccatagcccg 6780

caggtcgacc cgctgcgcga agagctggtc gcggcgctgg gagcgatccg gccgcgagcg 6840

gctgcggtgc cgatgcgctc gacggtgacg ggcggggtga ttgcgggtcc ggagctcgga 6900

gcgagctact gggcggacaa tcttcggcag ccggtgcgct tcgctgcggc ggcgcgagcg 6960

ctgctgggag gtggccccac gctgttcatc gagatgagcc cgcacccgat cctggtgccg 7020

cccctggacg agatccagac ggcggtcgag caagggggcg ctgcggtggg ctcgctgcgg 7080

cgagggcagg acgagcgcgc gacgctgctg gaggcgctgg ggacgctgtg ggcgtccggc 7140

tatccggtga gctgggctcg gctgttcccc gcgggcggca ggcgggttcc gctgccgacc 7200

tatccctggc agcacgagcg gtactggatc gaggacagcg tgcatggatc gaagccctcg 7260

ctgcggcttc ggcagcttcg caacggcgcc acggaccatc cgctgctcgg ggcttcattg 7320

ctcgtctcgg cgcgacccgg agctcacttg tgggagcaag cgctgagcga cgagaggctg 7380

tcctatcttt cggaacatag ggtccatggc gaagccgtgt tgccaagcgc ggcgtatgta 7440

gagatggcgc tcgccgccgg cgtagatctc tatggcacgg cgacgctggt gctggagcag 7500

ctggcgctcg agcgagccct cgccgtgcct tccgaaggcg gacgcatcgt gcaagtggcc 7560

ctcagcgaag aagggcccgg tcgggcctca ttccaggtat cgagtcgtga ggaggcaggt 7620

agaagctggg tgcggcacgc cacggggcac gtgtgtagcg accagagctc agcagtggga 7680

gcgttgaagg aagctccgtg ggagattcaa cagcgatgtc cgagcgtcct gtcgtcggag 7740

gcgctctatc cgctgctcaa cgagcacgcc ctcgactatg gtccctgctt ccagggtgtg 7800

gagcaggtgt ggctcggcac gggggaggtg ctcggccggg tacgcttgcc agaagacatg 7860

gcatcctcaa gtggcgccta tcggattcat cccgccttct tggatgcatg ttttcaagtg 7920

ctgaccgcgc tgctcaccac gccggaatcc atcgatattc ggaggcggct gacggatctc 7980

cacgaaccgg atctcccgcg gtccagggct ccggtgaatc aagcggtgag tgacacctgg 8040

ctgtgggacg ccgcgctgga cggtggacgg cgccagagcg cgagcgtgcc cgtcgagctg 8100

gtgctcggca gcttccatgc gaagtgggag gtcatggagc gcctcgcgca ggcgtacatc 8160

atcgacactc tccgcatatg ggacgtcttc tgcgctgctg gagagcgtca cacgatagac 8220

gagttgctcg tcaggcttca aatctctgtc ggctacagga aggtcatcaa gcgatggatg 8280

gatcaccttg tcgcgatcgg cgtcctcgta ggggacggag agcattttgt gagctctcag 8340

ccgctgccgg agcctgattt ggcggcggtg ctcgaggagg ccgggagggt gttcgccgac 8400

ctcccagtcc tacttgagtg gtgcaagttt gccggggaac ggctcgcgga cgtattgacc 8460

ggtaagacgc tcgcgctcga gatcctcttc cctggtggct cgttcgatat ggcggagcga 8520

atctatcaag attcgcccat cgcccgttac tcgaacggca tcgtgcgcgg tgtcgtcgag 8580

tcggcggcgc gggtggtagc accgtcggga atgttcagca tcttggagat cggagcaggg 8640

acgggcgcga ccaccgccac cgtcctcccg gtgttgctgc ctgaccggac agagtaccat 8700

tttaccgatg tttctccgct cttccttgct cgtgcggagc aaaaatttcg agatcatcca 8760

ttcctgaagt atggcattct ggatatcgac caggagccag ctggccaggg atacgcacat 8820

cagaagttcg acgtcatcgt cgcggccaac gtcatccatg cgacccgcga tataagagcc 8880

acggcgaagc gtctcctgtc gttgctcgcg cccggaggcc ttctggtgct ggtcgagggc 8940

acagggcatc cgatctggtt cgatatcacc acgggattga ttgaggggtg gcagaagtac 9000

gaagatgatc ttcgtaccga ccatccgctc ctgcctgctc ggacctggtg tgacctcctg 9060

cgccgggtag gctttgcgga cgccgtgagt ctgccaggcg acggatctcc ggcggggatc 9120

ctcggacagc acgtgatcct ctcgcgcgcg ccgggcatag caggagccgc ctgtgacagt 9180

tccggtgagt cggcgaccga atcgccggcc gcgcgtgcag tacggcagga atgggccgat 9240

ggctccgctg acgtcgtcca tcggatggcg ttggagagaa tgtacttcca ccgccggccg 9300

ggccggcagg tctgggtcca cggtcgattg cgtaccggtg gagacgcgtt cacgaaggcg 9360

ctcgctggag atctgctcct gttcgacgac accgggcagg tcgtggcaga ggttcagggg 9420

cttcgcctgc cgcagctcga ggcttctgct ttcgcgccgc gggacccgcg ggaagagtgg 9480

ttgtacgcgt tggaatggca gcgcaaagac cctataccag aggctccggc agccgcgtct 9540

tcttcctccg cgggggcttg gctcgtgctg atggaccagg gcgggacagg cgctgcgctc 9600

gtatcgctgc tggaagggcg aggcgaggcg tgcgtgcgcg tcatcgtggg tacggaatac 9660

gcctgcctcg cgccggggct gtatcaagtc gatccggcgc agtcagatgg ctttcatacc 9720

ctgctccgcg atgcattcgg cgaggaccgg atttgtcgcg cggtagtgca tatgtggagc 9780

cttgatgcga cggcagcagg ggagaggacg acaggggagt cgcttcaggc cgatcaactc 9840

ctggggagcc tgagcgcgct ttctctggtg caggcgctgg tgcgccggag gtggcgcaac 9900

atgccgcgac tttggctctt gacccgcgcc gtgcatgcgg tgggcgcgga ggacgcagcg 9960

gcctcggtgg cgcaggcgcc ggtgtggggc ctcggtcgga cgctcgcgct cgagcatcca 10020

gagctgcggt gcacgctcgt ggacgtgaac ccggcgccgt ctccagagga cgcagctgca 10080

ctggcggtgg agctcggggc gagcgacaga gaggaccaga tcgcattgcg ctcggatggc 10140

cgctacgtgg cgcgcctcgt gcggagctcc ttttccggca agcctgctac ggatcgcggc 10200

atccgggcgg acggcagtta tgtgatcacc gatggcatgg ggagagtggg gctctcggtt 10260

gcgcaatgga tggtgatgca gggggcccgc catgtggtgc tcgtggatcg cggcggcgct 10320

tccgaggcct cccgggatgc cctccggtcc atggccgagg ctggcgcgga ggtgcagatc 10380

gtggaggccg acgtggctcg gcgcgacgat gtcgctcggc tcctctcgaa gatcgaaccg 10440

tcgatgccgc cgcttcgggg gatcgtgtac gtggacggga ccttccaggt cgactcctcg 10500

atgctggagc tggatgccca tcgcttcaag gagtggatgt atcccaaggt gctcggagcg 10560

tggaacctgc acgcgctgac cagggataga tcgctggact tcttcgtcct gtactcctcg 10620

ggcacctcgc ttctgggctt gcccggacag gggagccgcg ccgccggtga cgccttcttg 10680

gacgccatcg cgcatcaccg gtgtaggctg ggccttacag cgatgagcat caactgggga 10740

ttgctcttcg aagcatcatc gccggcgacc ccgaacgacg gcggagcacg gctcgaatac 10800

cgggggatgg aaggtctcac gctggagcag ggagcggcgg cgctcgggcg cttgctcgca 10860

caacccaggg cgcaggtagg ggtgatgcgg ctgaatctgc gccagtggct ggatttctat 10920

cccaatgcgg cccgattggc gctgtgggcg gagttgatga aggagcgtga ccgcgccgac 10980

cgaggcgcgt cgaacgcatc gaacctgcgc gaggcgctgc agagcggcag gcccgaagat 11040

cgtcagttga ttctggagaa gcacttgagc gagctgttgg ggcgggggct gcgccttccg 11100

ccggcgagga tcgagcggca cgtgccgttc agcaatctcg gcatggactc gctgataggc 11160

ctggagctcc gcaaccgcat cgaggccgcg ctcggcatca ccgtgccggc gaccctgcta 11220

tggacctacc ctaccgtagc agctctgagc gggaacttgc tagacattct gtttccgaac 11280

gccggcgcga cccacgctcc ggccaccgag cgggagaaga gcttcgagaa cgatgccgca 11340

gatctcgagg ctctgctggg tatgacggac gagcagaagg acgcgttgct cgccgaaaag 11400

ctggcgcagc tcgcgcagat cgttggtgag taa 11433

<210> 6

<211> 7320

<212> DNA

<213> Sorangium cellulosum

<400> 6

atggcgacca cgaatgccgg gaagcttgag catgcccttc tgctcatgga caagcttgcg 60

aaaaagaacg cgtctttgga gcaagagcgg accgagccga tcgccatcat aggcattggc 120

tgccgcttcc ccggcggagc ggacactccg gaggcattct gggagctgct cgactcaggc 180

cgagacgcgg tccagccgct cgaccggcgc tgggcgctgg tcggggtcca tcccagtgag 240

gaggtgccgc gctgggccgg actgctcacc gaggcggtgg acggcttcga cgccgcgttc 300

tttggcacct cgcctcggga ggcgcggtcg ctcgatcctc agcaacgtct gctgctggag 360

gtcacctggg aagggctcga ggacgccggc atcgcacccc ggtccctcga cggcagccgc 420

accggggtat tcctgggcgc atgcagcagc gactactcgc ataccgttgc gcaacagcgg 480

cgcgaggagc aggacgcgta cgacatcacc ggcaatacgc tcagcgtcgc cgccggacgg 540

ttgtcttata cgctagggct gcagggaccc tgcctgaccg tcgacacggc ctgctcgtcg 600

tcgctcgtgg ccatccacct tgcctgccgc agcctgcgcg ctcgcgagag cgatctcgcg 660

ctggcgggag gcgtcaacat gctcctttcg tccaaagacg tgataatgct ggggcgcatc 720

caggcgctgt cgcccgatgg ccactgccgg acattcgacg cctcggccaa cgggttcgtc 780

cgtggggagg gctgcggtat ggtcgtgctc aaacggctct ccgacgccca gcgacatggc 840

gatcggatct gggctctgat ccggggttcg gccatgaatc aggatggccg gtcgacaggg 900

ttgatggcac ccaatgtgct cgctcaggag gcgctcttgc gcgaggcgct gcagagcgct 960

cgcgtcgacg ccggggccat tggttatgtc gagacccacg gaacggggac ctcgctcggc 1020

gacccgatcg aggtcgatgc gctgcgcgcc gtgatggggc cggcgcgggc cgatgggagc 1080

cgctgcgtgc tgggcgcagt gaagaccaac ctcggccacc tggagggcgc tgcaggcgtg 1140

gcgggtttga tcaaggcggc gctggctctg caccacgaac tgatcccgcg aaacctccat 1200

ttccacacgc tcaatccgcg gatccggatc gaggggaccg cgctcgcgct ggcgacggag 1260

ccggtgccgt ggccgcgggc gggccgaccg cgcttcgcgg gggtgagcgc gttcggcctc 1320

agcggcacca acgtccatgt cgtgctggag gaggcgccgg ccacggtgct cgcaccggcg 1380

acgccggggc gctcagcaga gcttttggtg ctgtcggcga agagcgccgc cgcgctggac 1440

gcacaggcgg cgcggctctc agcgcacatc gccgcgtacc cggagcaggg cctcggagac 1500

gtcgcgttca gcctggtagc gacgcgtagc ccgatggagc accggctcgc ggtggcggcg 1560

acctcgcgcg aggcgctgcg aagcgcgctg gaggttgcgg cgcaggggca gaccccggca 1620

ggcgcggcgc gcggcagggc cgcttcctcg cccggcaagc tcgccttcct gttcgccggg 1680

cagggcgcgc aggtgccggg catgggccgt gggttgtggg aggcgtggcc ggcgttccgc 1740

gagaccttcg accggtgcgt cacgctcttc gaccgggagc tccatcagcc gctctgcgag 1800

gtgatgtggg ccgagccggg cagcagcagg tcgtcgttgc tggaccagac ggcgttcacc 1860

cagccggcgc tctttgcgct ggagtacgcg ctggccgcgc tcttccggtc gtggggcgtg 1920

gagccggagc tcgtcgctgg ccatagcctc ggcgagctgg tggccgcctg cgtggcgggt 1980

gtgttctccc tcgaggacgc cgtgcgcttg gtggttgcgc gcggccggtt gatgcaggcg 2040

ctgccggccg gcggtgcgat ggtatcgatc gccgcgccgg aggccgacgt ggctgccgcg 2100

gtggcgccgc acgcagcgtc ggtgtcgatc gcggcagtca atgggccgga gcaggtggtg 2160

atcgcgggcg ccgagaaatt cgtgcagcag atcgcggcgg cgttcgcggc gcggggggcg 2220

cgaaccaaac cgctgcatgt ctcgcacgcg ttccactcgc cgctcatgga tccgatgctg 2280

gaggcgctcc ggcgggtggc ggagtcggtg acgtatcggc ggccttcgat ggcgctggtg 2340

agcaacctga gcgggaagcc ctgcacggat gaggtgtgcg cgccgggtta ctgggtgcgt 2400

cacgcgcgag aggcggtgcg cttcgcggac ggcgtgaagg cgctgcacgc ggccggtgcg 2460

ggcatcttcg tcgaggtggg cccgaagccg gcgctgctcg gccttttgcc ggcctgcctg 2520

ccggatgcca ggccggtgct gctgccagcg tcgcgcgccg ggcgtgacga ggctgcgagc 2580

gcgctggagg cgctgggtgg gttctgggtc gtcggtggat cggtcacctg gtcgggagtc 2640

ttcccttcgg gcggacggcg ggtaccgctg ccaacctatc cctggcagcg cgagcgttac 2700

tggatcgaag cgccggtcga tggtgaggcg gacggcatcg gccgtgctca ggcggggggc 2760

cacccccttc tgggtgaagt cttttccgtg tcgacccatg ccgatctgcg cctgtgggag 2820

acgacgctgg accgaaagcg gctgccgtgg ctcggcgagc accgggcgca gggggaggtc 2880

gtgtttcctc gcgccgggta cctggagatg gcgctgtcgt cgggggccga gatcttgggc 2940

gatggaccga tccaggtcac ggatgtggtg ctcatcgaga cgctgacctt cgcgggcgat 3000

acggcggtac cggtccaggt ggtgacgacc gaggagcgac cgggacggct acggttccag 3060

atagcgagtc gggggccggg tgaacgtcgc gcgtccttcc ggatccacgc ccgcggcgtg 3120

ctgcgccggg tcgggcgcgc cgagaccccg gcgaggttgg acctcgccgc cctgcgcgcc 3180

cggcttcatg ccgccgtgcc cgctgcggct acctatgggg cgctcgccga gatggggctt 3240

cgatacggcc cggcgttgcg ggggctcgcc gagctgtggc ggggtgaggg cgaggcgctg 3300

ggcagggtga gactgcctga ggccgccggc tccgccacag cctaccagct gcatccggtg 3360

ctgctggacg cgtgcgtcca aatgattgtt ggcgcgttcg ccgatcgcga tgaggcggcg 3420

ccgtgggcgc cggtggaggt gggctcggtg cggctgttcc agcggtctcc tggggagcta 3480

tggtgccatg cgcgcgtcgt gagcgatggt caacaggccc ccagccggtg gagcgccgac 3540

tttgagttga tggacggtac gggcgcggtg gtcgccgagg tctccgggct ggtggtggag 3600

cggcttgcga gcggtgtacg ccggcgcgaa gcagacgact ggttcctgga gctggattgg 3660

gagcccgcgg cgctcggtgg gcccaagatc acagccggcc ggtggctgct gctcggcgag 3720

ggtggcgggc tcgggctctc gttgtgctca gcgctgaagg ccgccggcca tgtcgtcgtg 3780

cacgccacgg gggacgacac gagcgccgca ggaatgcgcg cgctcctggc caacgcgttc 3840

gacggccagg ccccgacggc cgtggtgcac ctcagcagcc tcgacggggg cggccagctc 3900

gacccggggc tcggggcgca gggcgcgctc gacgcgcccc ggagcccaga tgtcgatgcc 3960

gatgccctcg agtcggcgct gatgcgtggc tgcgacagcg tgctctccct ggtgcaagcc 4020

ctggtcggca tggacctccg aaatgcgccg cggctgtggc tcttgacccg cggggctcag 4080

gcggccgccg ccggcgacgt ctccgtggtg caagcgccgc tgttggggct gggccgcacc 4140

atcgccttgg agcacgccga gctgcgctgt atcagcgtcg acctcgatcc agcccagcct 4200

gaaggggaag ccgatgcttt gctggccgag ctacttgcag atgatgccga ggaggaggtc 4260

gcgctgcgcg gtggcgagcg gtttgttgcg cggctcgtcc accggctgcc cgacgctcag 4320

cgccgggaga aggtcgagcc cgccggtgac aggccgttcc ggctagagat cgatgaaccc 4380

ggcgcgctgg accaactggt gctccgggcc acggggcggc gcgctcctgg tccgggcgag 4440

gtcgagatcg ccgtcgaagc ggcggggctc gactcgatcg acatccagct ggcgttgggc 4500

gttgctccca atgacctgcc tggagaagaa atcgagccgt cggtgctcgg acgcgagtgc 4560

gccgggcgca tcgtcgctgt gggcgagggc gtgaacggcc ttgtggtggg ccagccggtg 4620

atcgcccttg cggcgggagt atttgctacc catgtcacca cctcggccac gctggtgttg 4680

cctcggcctc tggggctctc ggcgaccgag gcggccgcga tgcccctcgc gtatttgacg 4740

gcctggtacg ccctcgacaa ggtcgcccac ctgcaggcgg gggagcgggt gctgatccat 4800

gcggaggccg gtggtgtcgg cctctgcgcg gtgcgatggg cgcagcgcgt gggcgccgag 4860

gtgtatgcga ccgccgacac gcccgagaaa cgtgcctacc tggcgtcgct gggcgtgcgg 4920

tacgtgagcg attcccgctc gggccggttc gccgcagacg tgcatgcatg gacggacggc 4980

gagggtgtgg acgtcgtgct cgactcgctt tcgggcgagc acatcgacaa gagcctcatg 5040

gtcctgcgcg cctgtggtcg ccttgtgaag ctgggcaggc gcgacgactg ccccgacacg 5100

cagcctgggc tgccgccgct cctacggaat ttttccttct cgcaggtgga cttgcgggga 5160

atgatgctcg atcaaccggc gaggatccgt gcgctcctcg acgagctgtt cgggttggtc 5220

gcagccgatc ccatcagccc actggggtgg gggttgcgcg ttggcggatc cctcacgcca 5280

ccgccggtcg agaccttccc gatctctcgc gcagccgagg cattccggag gatggcgcaa 5340

agacagcatc tcgggaagct cgtgctcacg ctggacgacc cggaggtgcg gatccgcgct 5400

ccggccgaat ccagcgtcgc cgtccgcgcg gacggcacct accttgtgac cggcggtctg 5460

ggtgggctcg gtctgcgcgt ggccggatgg ctggccgagc ggggcgcggg gcaactggtg 5520

ttggtgggcc gctccggtgc ggcgagcgca gagcagcgag ccgccgtggc ggcgctggag 5580

gcccacggcg cgcgcgtcac ggtggcgaaa gcggacgtcg ccgatcggtc acagatcgag 5640

cgggtcctcc gcgaggttac cgcgtcgggg atgccgctgc ggggtgtcgt gcatgcggca 5700

ggtcttgtgg atgacgggct gctgatgcag cagactccgg cgcggttccg cacggtgatg 5760

ggacctaagg tccagggagc cttgcacttg cacacgctga cacgcgatgc gcctctttcc 5820

ttcttcgtgc tgtacgcttc tgcagctggg ctgttcggct cgccaggcca gggcaactat 5880

gccgcagcca acgctttcct cgacgccctt tcgcatcacc gaagggcgca gggcctgccg 5940

gcgctgagca tcgactgggg catgttcacg gaggtgggga tggccgttgc gcaagcaaac 6000

cgtggcgcgc ggctgatctc tcgcgggatg cggggcatca cccccgatga ggggctgtcc 6060

gctctggcgc gcttgctcga gggtgatcgc gtgcagacgg gggtgatacc gatcactccg 6120

cgccagtggg tggagttcta cccggcaacg gcggcctcac ggaggttgtc gcggctggtg 6180

accacgcagc gcgcggtcgc tgatcggacc gccggggatc gggacctgct cgaacagctt 6240

gcctcggctg agccgagcgc gcgggcgggg ctgctgcagg acgtcgtgcg cgtgcaggtc 6300

tcgcatgtgc tgcgtctccc tgaaggcaag atcgaggtgg atgccccgct ctcgagcatg 6360

ggcatggact cgctgatgag cctggagctg cgcaaccgca tcgaggctgc gctgggcgtc 6420

gccgcgcctg cagccttggg gtggacgtac ccaacggtag cagcgataac gcgctggctg 6480

ctcgacgacg ccctcgtcgt ccggcttggc ggcgggtcgg acacggacga atcgacggca 6540

agcgccggtt cgttcgtcca cgtcctccgc tttcgtcctg tcgttaagcc gcgggctcgt 6600

ctcttctgct ttcacggttc tggcggctcg cccgagggct tccgttcctg gtcggagaag 6660

cctgagtgga gcgatctgga aatcgtggcc atgtggcacg atcgcagcct cgcctccgag 6720

gacacgcctg gtaagaagta cgtccaagag gcggcctcgc tgattcagca ctatgcagac 6780

gcaccgtttg cgttagtagg gttcagcctg ggtgtccggt tcgtcatggg gacagccgtg 6840

gagctcgcca gtcgttccgg cgcaccggct ccgctggccg ttttcacgtt gggcggcagc 6900

ttgatctctt cttcagagat cgccccggag atggagaccg acataatagc caagctcttc 6960

ttccgaaatg ccgcgggttt cgtgcgatcc acccaacaag ttcaggccga tgctcgcgca 7020

gacaaggtca tcacagacac gatgatggct ccggcccccg gggattcgaa ggagccgccc 7080

gtgaagatcg cggtccctat cgtcgccatc gccggctcgg acgatgtgat cgtgcctcca 7140

agcgacgttc aggatctaca atctcgcacc acggagcgct tctatatgca tctccttccc 7200

ggagatcacg agtttctcgt cgatcgaagt cgcgagatca tgcacatcgt cgactcgcat 7260

ctcaatccgc tgctcgccgc gaagacgacg tcgtcaggcc cggcgttcga ggcaaaatga 7320

<210> 7

<211> 2179

<212> DNA

<213> Artificial sequence

<220>

<223> PCR targeting fragment 1

<400> 7

ccctgctgtg gacctacccc acggtggcag cgctgagtgc gcatctggct agcttggctg 60

agccattcga gtgctgggtt gttgtctctg gacactgatc catgggaaac tactcagcac 120

catctctagt cgacctgcag gcatgcaagc ttcgattggc taggtctagc ggagtgtata 180

ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa 240

aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc 300

tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg 360

gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc 420

aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa 480

tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc 540

cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc 600

gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg 660

actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc 720

ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat 780

ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt 840

ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc 900

ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga 960

ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta gagtcgacct 1020

gcagcggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 1080

ttatcaaaaa ggatcttcac ctagatcctt ttcgaccgaa taaatacctg tgacggaaga 1140

tcacttcgca gaataaataa atcctggtgt ccctgttgat accgggaagc cctgggccaa 1200

cttttggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga 1260

aataagatca ctaccgggcg tattttttga gttgtcgaga ttttcaggag ctaaggaagc 1320

taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa 1380

agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct 1440

ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt 1500

tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttacgtatgg caatgaaaga 1560

cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac 1620

tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat 1680

atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat 1740

tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa 1800

cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca 1860

aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt 1920

ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc 1980

gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggttgctacg cctgaattcg 2040

agctcggtac ccggggatcc tctagtcgac gatgaccagg tttttgacga aagtgatcca 2100

gatgatccag ctctacactg gttcatgtgc cccagggcct gatcccttcg accgacggct 2160

tcgcctggca acgcggata 2179

<210> 8

<211> 2488

<212> DNA

<213> Artificial sequence

<220>

<223> PCR targeting fragment 2

<400> 8

gggcgatcgc cgcgaagaag gcctccagct ccggcgtcgg gatgcgacca ttggctgagc 60

cattcgagtg ctgggttgtt gtctctggac accgatccat gggaaactac tcagcaccat 120

ctctagtcga cctgcaggca tgcaagcttc gattggctag gtctagcgga gtgtatactg 180

gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa 240

aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca 300

ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg 360

gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa 420

gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca 480

gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct 540

cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg 600

tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact 660

gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg 720

agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta 780

gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt 840

gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc 900

gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca 960

aaacgatctc aagaagatca tcttattaat cagataaaat atttctagag tcgacctgca 1020

gctaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 1080

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 1140

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 1200

tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 1260

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 1320

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 1380

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 1440

gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg 1500

ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac 1560

atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg 1620

acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa 1680

ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt 1740

atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc 1800

actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa 1860

aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat 1920

tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac 1980

ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc 2040

tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat 2100

ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga 2160

cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag 2220

ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg 2280

aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcga 2340

attcgagctc ggtacccggg gatcctctag agattgacca ggtttttgac gaaactgatc 2400

cagatgatcc agctctacac tggttcatgt gcgctagcca tgtcgtctct acgggggatg 2460

gggaatccgt gcgcccgcct gatacagg 2488

<210> 9

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> attB0

<400> 9

ggcttgtcga cgacggcggt ctccgtcgtc aggatcat 38

<210> 10

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> attP0

<400> 10

ggtttgtctg gtcaaccacc gcggtctcag tggtgtacgg tacaaacc 48

<210> 11

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> attB6

<400> 11

ggcttgtcga cgacggcgct ctccgtcgtc aggatcat 38

<210> 12

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> attP6

<400> 12

ggtttgtctg gtcaaccacc gcgctctcag tggtgtacgg tacaaacc 48

<210> 13

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> attB7

<400> 13

ggcttgtcga cgacggcgaa ctccgtcgtc aggatcat 38

<210> 14

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> attP7

<400> 14

ggtttgtctg gtcaaccacc gcgaactcag tggtgtacgg tacaaacc 48

<210> 15

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> attB13

<400> 15

ggcttgtcga cgacggcgca ctccgtcgtc aggatcat 38

<210> 16

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> attP13

<400> 16

ggtttgtctg gtcaaccacc gcgcactcag tggtgtacgg tacaaacc 48

<210> 17

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> attB15

<400> 17

ggcttgtcga cgacggcgcc ctccgtcgtc aggatcat 38

<210> 18

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> attP15

<400> 18

ggtttgtctg gtcaaccacc gcgccctcag tggtgtacgg tacaaacc 48

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> BSD-F

<400> 19

ctcgcggggg tatcgcttcc 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> BSD-R

<400> 20

tcagccaatc gactggcgag 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> accA1-F

<400> 21

atgcgcaagg tgctcatcgc 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> accA1-R

<400> 22

tcagtccttg atctcgcaga 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> pccA-F

<400> 23

atgatcactt ccgtcctcgt 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> pccA-R

<400> 24

tcagtcggac tcgacgaccg 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> pccB-F

<400> 25

atgtccgagc cggaagagca 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> pccB-R

<400> 26

ttacaggggg atgttgccgt 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> epi-F

<400> 27

atgctgacgc gaatcgacca 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> epi-R

<400> 28

tcagtgctca ggtgactcaa 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> MatB-F

<400> 29

atgtcctctc tcttcccggc 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> MatB-R

<400> 30

tcagtcacgg ttcagcgccc 20

<210> 31

<211> 1773

<212> DNA

<213> Streptomyces coelicolor A3(2)

<400> 31

atgcgcaagg tgctcatcgc caatcgtggc gaaatcgctg tccgcgtggc ccgggcctgc 60

cgggacgccg ggatcgcgag cgtggccgtc tacgcggatc cggaccggga cgcgttgcac 120

gtccgtgccg ctgatgaggc gttcgccctg ggtggtgaca cccccgcgac cagctatctg 180

gacatcgcca aggtcctcaa agccgcgcgc gagtcgggcg cggacgccat ccaccccggc 240

tacggattcc tctcggagaa cgccgagttc gcgcaggcgg tcctggacgc cggcctgatc 300

tggatcggcc cgcccccgca cgccatccgc gacctgggcg acaaggtcgc cgcccgccac 360

atcgcccagc gggccggcgc ccccctggtc gccggcaccc ccgaccccgt ctccggcgcg 420

gacgaggtcg tcgccttcgc caaggagcac ggcctgccca tcgccatcaa ggccgccttc 480

ggcggcggcg ggcgcggcct caaggtcgcc cgcaccctcg aagaggtgcc ggagctgtac 540

gactccgccg tccgcgaggc cgtggccgcc ttcggccgcg gggagtgctt cgtcgagcgc 600

tacctcgaca agccccgcca cgtggagacc cagtgcctgg ccgacaccca cggcaacgtg 660

gtcgtcgtct ccacccgcga ctgctccctc cagcgccgcc accaaaagct cgtcgaggag 720

gcccccgcgc ccttcctctc cgaggcccag acggagcagc tgtactcatc ctccaaggcc 780

atcctgaagg aggccggcta cgtcggcgcc ggcaccgtgg agttcctcgt cggcatggac 840

ggcacgatct ccttcctgga ggtcaacacc cgcctccagg tcgagcaccc ggtcaccgag 900

gaagtcgccg gcatcgacct ggtccgcgag atgttccgca tcgccgacgg cgaggaactc 960

ggctacgacg accccgccct gcgcggccac tccttcgagt tccgcatcaa cggcgaggac 1020

cccggccgcg gcttcctgcc cgcccccggc accgtcaccc tcttcgacgc gcccaccggc 1080

cccggcgtcc gcctggacgc cggcgtcgag tccggctccg tcatcggccc cgcctgggac 1140

tccctcctcg ccaaactgat cgtcaccggc cgcacccgcg ccgaggcact ccagcgcgcg 1200

gcccgcgccc tggacgagtt caccgtcgag ggcatggcca ccgccatccc cttccaccgc 1260

acggtcgtcc gcgacccggc cttcgccccc gaactcaccg gctccacgga ccccttcacc 1320

gtccacaccc ggtggatcga gacggagttc gtcaacgaga tcaagccctt caccacgccc 1380

gccgacaccg agacggacga ggagtcgggc cgggagacgg tcgtcgtcga ggtcggcggc 1440

aagcgcctgg aagtctccct cccctccagc ctgggcatgt ccctggcccg caccggcctg 1500

gccgccgggg cccgccccaa gcgccgcgcg gccaagaagt ccggccccgc cgcctcgggc 1560

gacaccctcg cctccccgat gcagggcacg atcgtcaaga tcgccgtcga ggaaggccag 1620

gaagtccagg aaggcgacct catcgtcgta ctcgaggcga tgaagatgga acagcccctc 1680

aacgcccaca ggtccggcac catcaagggc ctcaccgccg aggtcggcgc ctccctcacc 1740

tccggcgccg ccatctgcga gatcaaggac tga 1773

<210> 32

<211> 1845

<212> DNA

<213> Streptomyces coelicolor A3(2)

<400> 32

atgatcactt ccgtcctcgt cgccaaccgc ggcgagatcg cctgccgcgt cttcagcacc 60

tgccgcgagt cgggcatccg caccgtcgcc gtgcactcgg acgccgacgc gaacgccctc 120

cacgcgcgcg tggccgacgc cgccgtacgc ctgccgggcg cggcccccgc cgacacctat 180

ctgcgcggcg acctgatcgt gaaggccgcc gtcgccgccg gagccgacgc cgtccacccc 240

ggctacggct tcctctccga gaacgccgac ttcgcgcgcg ccgtacggga cgcggggctg 300

gtgtggatcg gaccgccgcc cgaggccatc gaggcgatgg cgtccaagac ccgcgccaag 360

gaactgatgg gcatcgcgcc cctcaccgac gtcaccgagg ccgacctgcc ggtgctggtg 420

aaggcggcgg cgggcggcgg cggacgcggc atgcgcgtcg tacgccgcct cgccgacctc 480

gacgccgaac tgaccgccgc ccgcgcggag gccgcgagcg ccttcggcga cggcgaggtc 540

ttcgtcgagc cgtatgtggt cgacggccgc cacgtcgagg tgcagatcct cgccgacacc 600

cacggcacgg tgtgggtgct cggcacccgc gactgctccc tccaacgccg ccaccagaag 660

gtgatcgagg aggccccggc gcccggcctg acccccgggc tcaccgccga actccacgac 720

ctcgccgtgc gcgccgcccg cgccgtcgac tacgtcggcg cgggcaccgt cgagttcctc 780

gtcgccgacg gcacggcgca cttcctggag atgaacaccc gcctccaggt cgaacacccg 840

gtcacggagg cggtcttcgg catcgacctc gtcgccctcc agctccggat cgccgaaggc 900

cacgccctcg acgacgaccc cccgcgcgcg cgtggccacg ccgtcgaggc ccgcctctac 960

gccgaggacc cggcgaacgg ctgggccccg cagaccggcc gcctgcaccg cctcgccgtg 1020

ccggacggca tccgcctgga caccggctac accggcggcg acgacatcgg cgtccactac 1080

gacccgatgc tcgccaaggc ggtcgcccac gcacccacgc gcgcggaggc cgtccgccga 1140

ctcgccggcg ccctggaacg cgccgcgatc cacggcccgg tcaccaaccg cgacctcctc 1200

gtccgctccc tgcgccacga ggagttcacc tccggccgca tggacacggg cttctacgac 1260

cgccacctcg ccgccctcac cgagccggcc cccgaccccc tcgccccgct ggccgccgcc 1320

ctcgccgacg cgagcacccg tgcgggacgc ttcggcggct ggcgcaacct gccctcgcaa 1380

ccgcaggtca agcggtacgc cgtggcgggc gaggaacacg aggtccgtta cgggcacacc 1440

cgcacgggcc tcaccgccga gggcgtccgc gtcgtccacg cgggccccga ccgggtcgtc 1500

ctcgaagcgg acggcgtaca acgccccttc gacatcgccc gctacggcga ccacgtgcac 1560

gtcaacacca cgcgcctcac cgccctgccc cgcttccccg accccaccac ccagcacgcc 1620

cccggctccc tcctggcccc catgccgggc acggtcgtcc gcgtcgcgga gggcctgacc 1680

gagggcacca ccgtccaggc gggccagccg ttgctgtggc tggaggccat gaagatggaa 1740

cacaggatca ccgccccggt gacagggagg ctgaccgcac tcccggcggg cctcggacga 1800

caagtagaga tgggcgccct cttggcggtc gtcgagtccg actga 1845

<210> 33

<211> 1593

<212> DNA

<213> Streptomyces coelicolor A3(2)

<400> 33

atgtccgagc cggaagagca gcagcccgac atccacacga ccgcgggcaa gctcgcggat 60

ctcaggcgcc gtatcgagga agcgacgcac gccggttccg cacgcgccgt cgagaagcag 120

cacgccaagg gcaagctgac ggctcgtgaa cgcatcgacc tcctcctcga cgagggttcc 180

ttcgtcgagc tggacgagtt cgcccggcac cgctccacca acttcggcct cgacgccaac 240

cgcccctacg gcgacggcgt cgtcaccggc tacggcaccg tcgacggccg ccccgtggcc 300

gtcttctccc aggacttcac cgtcttcggc ggcgcgctgg gcgaggtcta cggccagaag 360

atcgtcaagg tgatggactt cgccctcaag accggctgcc cggtcgtcgg catcaacgac 420

tccggcggcg cccgcatcca ggagggcgtg gcctccctcg gcgcctacgg cgagatcttc 480

cgccgcaaca cccacgcctc cggcgtgatc ccgcagatca gcctggtcgt cggcccgtgt 540

gcgggcggcg cggtgtactc ccccgcgatc accgacttca cggtgatggt ggaccagacc 600

agccacatgt tcatcaccgg tcccgacgtc atcaagacgg tcaccggcga ggacgtcggc 660

ttcgaggagc tgggcggcgc ccgcacccac aactccacct cgggcgtggc ccaccacatg 720

gccggcgacg agaaggacgc ggtcgagtac gtcaagcagc tcctgtcgta cctgccgtcc 780

aacaacctct ccgagccccc cgccttcccg gaggaggcgg acctcgcggt cacggacgag 840

gacgccgagc tggacacgat cgtcccggac tcggcgaacc agccctacga catgcactcc 900

gtcatcgagc acgtcctgga cgacgccgag ttcttcgaga cgcaacccct cttcgcgccg 960

aacatcctca ccggcttcgg ccgcgtggag ggccgcccgg tcggcatcgt cgccaaccag 1020

cccatgcagt tcgccggctg cctggacatc acggcctccg agaaggcggc ccgcttcgtg 1080

cgcacctgcg acgccttcaa cgtccccgtc ctcaccttcg tggacgtccc cggcttcctg 1140

cccggcgtcg accaggagca cgacggcatc atccgccgcg gcgccaagct gatcttcgcc 1200

tacgccgagg ccacggtgcc gctcatcacg gtcatcaccc gcaaggcctt cggcggcgcc 1260

tacgacgtca tgggctccaa gcacctgggc gccgacctca acctggcctg gcccaccgcc 1320

cagatcgccg tcatgggcgc ccaaggcgcg gtcaacatcc tgcaccgccg caccatcgcc 1380

gacgccggtg acgacgccga ggccacccgg gcccgcctga tccaggagta cgaggacgcc 1440

ctcctcaacc cctacacggc ggccgaacgc ggctacgtcg acgccgtgat catgccctcc 1500

gacactcgcc gccacatcgt ccgcggcctg cgccagctgc gcaccaagcg cgagtccctg 1560

cccccgaaga agcacggcaa catccccctg taa 1593

<210> 34

<211> 441

<212> DNA

<213> Streptomyces coelicolor A3(2)

<400> 34

atgctgacgc gaatcgacca catcggaatc gcctgccacg acctcgacgc gaccgtcgag 60

ttctaccgtg ccacctacgg cttcgaggtg ttccacaccg aggtcaacga ggagcagggg 120

gtgcgcgagg ccatgctcaa gatcaacgat acgtcggacg ggggcgcctc gtacctccag 180

ctcctggagc cgacccgcga ggactccgcg gtcggcaagt ggctcgcgaa gaacggcgag 240

ggcgtccacc acatcgcctt cggtacggcg gacgtggacg cggacgccgc ggacatccgc 300

gacaagggcg tacgcgttct gtacgacgag ccccggcgcg gttccatggg gtcgcggatc 360

accttcctgc accccaagga ctgccatggc gtactgacag aactggtcac ttcggcggcc 420

gttgagtcac ctgagcactg a 441

<210> 35

<211> 1458

<212> DNA

<213> Streptomyces coelicolor A3(2)

<400> 35

atgtcctctc tcttcccggc cctctccccg gccccgaccg gcgccccggc cgaccggccc 60

gcgctgcggt tcggcgagcg ctccctgacc tacgcggaac tcgccgcggc ggcgggcgcc 120

acggccgggc ggatcggcgg cgccggccgg gtcgcggtct gggccacccc ggcgatggag 180

accggcgtcg ccgtggtggc ggcgctgctg gccggggtcg ccgccgtacc gctcaacccg 240

aagtccggcg acaaggaact cgcgcacatc ctctccgaca gcgcgccctc gctcgtcctg 300

gcgcccccgg acgcggaact cccgcccgcc ctcggggccc tggagcgcgt cgacgtcgac 360

gtgcgggccc gcggggcggt ccccgaggac ggtgccgacg acggcgaccc cgcgctcgtc 420

gtctacacct cgggcaccac gggaccgccg aagggcgccg tcatcccccg gcgggcgctc 480

gccacgaccc tggacgcgct cgccgacgcg tggcagtgga ccggcgagga cgtgctggtg 540

caggggctgc cgctgttcca cgtgcacggg ctggtcctcg gcatcctcgg cccgctgcgc 600

cggggcgggt ccgtgcggca cctgggccgg ttctccaccg agggtgcggc gcgggagctg 660

aacgacggcg cgaccatgct gttcggggtg ccgacgatgt accaccggat cgccgagacg 720

ctccccgccg acccggagct ggcgaaggcg ctcgccgggg cccggctgct ggtgtcgggg 780

tcggccgcgc tgccggtgca cgaccacgag cgcatcgccg ccgccaccgg gcgccgggtg 840

atcgagcggt acggcatgac cgagacgctg atgaacacca gcgtgcgcgc cgacggcgag 900

ccgcgcgccg ggacggtggg cgtgccgctg cccggtgtgg agctgcggct ggtggaggag 960

gacggcacgc cgatcgcggc gctcgacggg gagagcgtcg gcgagatcca ggttcgcggc 1020

ccgaacctgt tcaccgagta cctgaaccgc cccgacgcca ccgccgccgc cttcaccgag 1080

gacggcttct tccgcaccgg cgacatggcg gtgcgcgacc ccgacggcta tgtccgcatc 1140

gtcggccgca aggccaccga cctgatcaag agcggcggtt acaagatcgg ggccggggag 1200

atcgagaacg ccctgctcga acacccggag gtccgggagg ccgccgtcac cggcgaaccc 1260

gaccccgacc tcggggaacg gatcgtggcc tggatcgtcc cggccgaccc cgccgccccg 1320

cccgccctcg gcacgctggc cgaccacgtc gccgcccggc tcgccccgca caagcggccg 1380

cgcgtcgtcc ggtacctcga cgcggtgccc cgcaacgaca tggggaagat catgaagcgg 1440

gcgctgaacc gtgactga 1458

<210> 36

<211> 73

<212> DNA

<213> Myxococcus xanthus (Myxococcus xanthus)

<400> 36

ggtcttgtag ctcaggggat agagcactcg gttgcggacc gagaggccgc aggttcgact 60

cctgccagga cca 73

<210> 37

<211> 74

<212> DNA

<213> Myxococcus xanthus (Myxococcus xanthus)

<400> 37

gcgttcgtag ctcaactgga tagagcaccg ggcttcgaac ccgggggttg ggggttcaag 60

tccctccgag cgcg 74

<210> 38

<211> 71

<212> DNA

<213> Myxococcus xanthus (Myxococcus xanthus)

<400> 38

tggggaatcg tctaacggca ggacagcaga ctctgactct gcttatctag gttcgaatcc 60

tagttcccca g 71

<210> 39

<211> 72

<212> DNA

<213> Myxococcus xanthus (Myxococcus xanthus)

<400> 39

ggccctgtcg tctagcggtt aggacggagc cctctcacgg ctcaaactcg ggttcgaatc 60

ccggcagggt ca

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> tRNA-F

<400> 40

cttctgacac cgcgcctcgt 20

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> tRNA-R

<400> 41

tgggcgtctc agtgtgaccc 20

<210> 42

<211> 200

<212> DNA

<213> Artificial sequence

<220>

<223> PKan

<400> 42

tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg 60

aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact 120

ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct gatcaagaga 180

caggatgagg atcgtttcgc 200

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> epo1A3-F

<400> 43

tttgctcaca tgttctttcc 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> epo1A3-R

<400> 44

ggtctgacgc tcagtggaac 20

<210> 45

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> epo3K5-F

<400> 45

ggaaagaaca tgtgagcaaa ggaagatgcc aggaagatac 40

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> epo3K5-R

<400> 46

gttccactga gcgtcagacc 20

<210> 47

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> epoA-F

<400> 47

gaattcgcgg ccgcttctag atggcggatc gtcccatcg 39

<210> 48

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> epoA-R

<400> 48

tgcagcggcc gctactagta tcatagggca atgatttccc agtc 44

<210> 49

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> epoB-F

<400> 49

gaattcgcgg ccgcttctag atgacgatca atcagcttct gaac 44

<210> 50

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> epoB-R

<400> 50

tgcagcggcc gctactagta ttagctacgt ctcctgccct tg 42

<210> 51

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> epoC-F

<400> 51

gaattcgcgg ccgcttctag atggaagaac aagattcctc cgctatcg 48

<210> 52

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> epoC-R

<400> 52

tgcagcggcc gctactagta tcatgtaagc gccttgaatt tag 43

<210> 53

<211> 74

<212> DNA

<213> Artificial sequence

<220>

<223> epoD-F

<400> 53

ggttgttcgc gttgattgat gagtcactcg cgcgtgcggg aaagaggtga tactagtagc 60

ggccgctgca gtcc 74

<210> 54

<211> 71

<212> DNA

<213> Artificial sequence

<220>

<223> epoD-R

<400> 54

gccgcttgtt tcagcggatt ctgctgtgcc gtaggaccgc gagtagtcat ctagaagcgg 60

ccgcgaattc c 71

<210> 55

<211> 74

<212> DNA

<213> Artificial sequence

<220>

<223> epoE-F

<400> 55

cgttgctcgc cgaaaagctg gcgcagctcg cgcagatcgt tggtgagtaa tactagtagc 60

ggccgctgca gtcc 74

<210> 56

<211> 71

<212> DNA

<213> Artificial sequence

<220>

<223> epoE-R

<400> 56

tctcggtctg tcacgcaatc acctctttcc cgcacgcgcg agtgactcat ctagaagcgg 60

ccgcgaattc c 71

<210> 57

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> epoF-F

<400> 57

gaattcgcgg ccgcttctag atggcgacca cgaatgccgg gaag 44

<210> 58

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> epoF-R

<400> 58

tgcagcggcc gctactagta tcattttgcc tcgaacgccg ggcctg 46

<210> 59

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> PKan-F

<400> 59

ggaattcgcg gccgcttcta gatagcttgc agtgggctta c 41

<210> 60

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> PKan-R

<400> 60

gactagtacg atcctcatcc tgtctcttg 29

<210> 61

<211> 495

<212> DNA

<213> Artificial sequence

<220>

<223> attP0-ccdB-attB15

<400> 61

ggtttgtctg gtcaaccacc gcggtctcag tggtgtacgg tacaaaccca aagcttcggt 60

tgcgcgctga tttgtgcggc ataagaatat atactgatat gtatacccga agtatgtccg 120

gaagaggtgt gctatgcagt tcaaggttta cacctataaa agagagagcc gctatcgcct 180

gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct 240

ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat 300

cggggatgaa agctggcgca tgatgaccac ccagatggtc agtgtgccgg tctccgtcat 360

cggagaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaatct 420

gatgttctgg ggaatataaa aggaaaaaag gatccccggc ttgtcgacga cggcgccctc 480

cgtcgtcagg atcat 495

<210> 62

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> ccdB-F

<400> 62

gtaaaacgac ggccagt 17

<210> 63

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> ccdB-R

<400> 63

tgctagttat tgctcagcgg 20

<210> 64

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> ccdB-vector-F

<400> 64

ggcactggcc gtcgttttac caatctgtac ctccttaagt c 41

<210> 65

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> ccdB-vector-R

<400> 65

ccgctgagca ataactagca ggtatcgctt cccgaacctc 40

<210> 66

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> BSD-ccdB-F

<400> 66

ctgaattggc tatccgcgtg caagagatta cgcgcagacc 40

<210> 67

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> BSD-ccdB-R

<400> 67

gaagagcaca tacctcagtc gcagctcacg gtaactgatg 40

<210> 68

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> ST-F

<400> 68

gactgaggta tgtgctcttc 20

<210> 69

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> ST-R

<400> 69

cacgcggata gccaattcag 20

<210> 70

<211> 611

<212> DNA

<213> Artificial sequence

<220>

<223> tRNA

<400> 70

cttctgacac cgcgcctcgt ggctggggcg ctctcgcagt cgcagttccg gtcttgtagc 60

tcaggggata gagcactcgg ttgcggaccg agaggccgca ggttcgactc ctgccaggac 120

cactcctcct tcagttgtct ggttcgatcc cggttggccg cccttcttta cccgccctcg 180

aaattgcgtt cgtagctcaa ctggatagag caccgggctt cgaacccggg ggttgggggt 240

tcaagtccct ccgagcgcgc accttccaag ttgttgcagt cccgaagtgg tattggtaga 300

gaagcgccgc ggtcgagcag gtccttgggg aatcgtctaa cggcaggaca gcagactctg 360

actctgctta tctaggttcg aatcctagtt ccccagcttg tagtcccgca gttgcagtgc 420

tcgttggtcc gaagcaacct gagcaggacg aaacaaaagg gttgacgagg gcagacgaaa 480

aaaggtagaa atcgcgggca gttagcggcg gttgaaacaa gtatcggccc tgtcgtctag 540

cggttaggac ggagccctct cacggctcaa actcgggttc gaatcccggc agggtcacac 600

tgagacgccc a 611

<210> 71

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<223> BSD-epo-F

<400> 71

ggaacatcga atcactcaac gtcatcttga ggccctccaa agctggataa ctcgcggggg 60

tatcgcttcc 70

<210> 72

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<223> BSD-epo-R

<400> 72

acgatcgcaa tcggatcttc ggctgcgcgc tcgatgggac gatccgccac aatctgtacc 60

tccttaagtc 70

<210> 73

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> epo-vector-F

<400> 73

tactagtagc ggccgctgca gtcc 24

<210> 74

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> epo-vector-R

<400> 74

ctagaagcgg ccgcgaattc cagaaatc 28

Claims (21)

1. A method for heterogeneously expressing epothilone is characterized in that an epothilone gene cluster is introduced into host bacteria, and meanwhile, an epothilone precursor synthesis way is supplemented;

the host bacterium is Burkholderia (Burkholderia) DSM7029 strain;

the precursor synthesis pathway is a synthesis pathway of S-methylmalonyl-coenzyme A; the synthesis pathway for supplementing the S-methylmalonyl coenzyme A comprises supplementing a PCC pathway, a MatB pathway and a mutase-isomerase pathway; the PCC pathway is complemented by the addition of propionyl-coa carboxylase; the MatB pathway is complemented by the addition of malonyl-coa/methylmalonyl-coa synthetase; the mutase-isomerase pathway is complemented by the addition of methylmalonyl-coa isomerase;

tRNA genes are also introduced into the host bacteria, and the tRNA genes are Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes.

2. The method of heterologously expressing an epothilone of claim 1, wherein the propionyl-coa carboxylase is the accA1/pccB or pccA/pccB gene of streptomyces coelicolor (s.coelicolor) a3 (2).

3. The method of heterologously expressing an epothilone of claim 1, wherein methylmalonyl-coa isomerase is the epi gene of streptomyces coelicolor (s.coelicolor) a3 (2).

4. The method of heterologously expressing an epothilone of claim 1, wherein the malonyl-coa/methylmalonyl-coa synthetase is the matB gene of streptomyces coelicolor a3 (2).

5. The method for the heterologous expression of an epothilone according to claim 1, wherein the argani-GCG, argani-TCG, Gln anti-CTG and gluani-CTC genes are derived from Myxococcus aurantii (Myxococcus xanthus) DK 1622.

6. The method of heterologously expressing an epothilone of claim 1 wherein a promoter sequence is added before one or more genes in the epothilone gene cluster.

7. The method of heterologously expressing an epothilone of claim 6 wherein a promoter sequence is added before one or more of the epoA, epoB, epoC, epoD, epoE and epoF 6 genes in the epothilone gene cluster.

8. The method of heterologously expressing an epothilone of claim 6, wherein a promoter sequence is added before each of the epoA, epoB, epoC, epoD, epoE, and epoF 6 genes in the epothilone gene cluster.

9. The method for the heterologous expression of an epothilone according to claim 7 or 8, wherein the promoter is PKan.

10. The method of heterologously expressing an epothilone of claim 9 wherein the promoter sequence is added by gene re-splicing.

11. The method of heterologously expressing an epothilone of claim 10 wherein the splicing is performed using the Bxb1 integrase splicing technique.

12. A genetic engineering strain for heterogeneously expressing epothilone is characterized in that an epothilone gene cluster is introduced into the genetic engineering strain and an epothilone precursor synthesis way is supplemented; wherein, the basic strain of the genetic engineering strain is Burkholderia (Burkholderia) DSM7029 strain; wherein the base strain is supplemented with the synthetic pathway of epothilone precursor S-methylmalonyl-CoA, including the PCC pathway, the MatB pathway and the mutase-isomerase pathway; the PCC pathway is complemented by the addition of propionyl-coa carboxylase; the MatB pathway is complemented by the addition of malonyl-coa/methylmalonyl-coa synthetase; the mutase-isomerase pathway is complemented by the addition of methylmalonyl-coa isomerase; tRNA genes are also added into the basic strain, and the tRNA genes are Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes.

13. The genetically engineered strain for the heterologous expression of epothilones according to claim 12, wherein in the base strain the PCC pathway is complemented by the addition of accA1/pccB of streptomyces coelicolor (s.coelicolor) a3 (2); supplementation of the mutase-isomerase pathway by addition of the epi gene of streptomyces coelicolor a3 (2); the matB pathway was supplemented by the addition of the matB gene of streptomyces coelicolor a3 (2).

14. The genetically engineered strain of heterologous expression of an epothilone of claim 12, wherein a promoter sequence is added before one or more genes of the epothilone gene cluster.

15. The genetically engineered strain of heterologous expression of an epothilone of claim 14, wherein a promoter sequence is added to each of the 6 epoA, epoB, epoC, epoD, epoE and epoF genes in the epothilone gene cluster.

16. The genetically engineered strain of heterologous expression of an epothilone according to claim 15, wherein the promoter sequence is added by splicing in order epoA, epoB, epoC, epoD, epoE to epoF by means of a re-splicing.

17. The genetically engineered strain of heterologous expression of an epothilone of claim 16, wherein the promoter is pka.

18. A genetic engineering strain for heterologous expression of epothilone is characterized in that the genetic engineering strain is Polysaccharum brevicornum (Polyangium brachysporum) MMR11 with the preservation number of CCTCC M2017037, and is preserved in the China center for type culture collection management in 2017, 1 month and 19 days.

19. A process for the production of epothilones, characterized in that a strain according to any of claims 12 to 17 is provided which is fermented at 30 ± 2 ℃ in a fermentation medium.

20. The method of claim 19, wherein the fermentation medium is CYMG fermentation medium formulated per liter as tyrosone 8g, yeast extract 4g, magnesium chloride hexahydrate 4.06g, 50% glycerol 10ml, trace elements 1ml, sodium acetate 50mg, sodium propionate 100mg, methylmalonic acid 100mg, cysteine 2.5mg, serine 5mg, XAD-16 macroporous adsorbent wet weight 1%, pH adjusted 7.0-7.5; wherein the trace elements are: 0.79g of manganese chloride tetrahydrate, 0.15g of zinc sulfate heptahydrate, 0.64g of copper sulfate pentahydrate and 0.11g of ferrous sulfate heptahydrate are dissolved in 100mL of water.

21. The method of producing an epothilone of claim 19 wherein the fermentation time is 3 days.

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